U.S. patent application number 15/643577 was filed with the patent office on 2017-10-26 for simplified device for nucleic acid amplification adn method for using same.
The applicant listed for this patent is bioMerieux, S.A.. Invention is credited to Patrick Broyer, Laurent Drazek, Agnes Dupont Filliard, Michel Guy, Thierry Kollaroczy, Frederic Pinston, Magaly Ponsard-Fillette.
Application Number | 20170304816 15/643577 |
Document ID | / |
Family ID | 42135587 |
Filed Date | 2017-10-26 |
United States Patent
Application |
20170304816 |
Kind Code |
A1 |
Broyer; Patrick ; et
al. |
October 26, 2017 |
SIMPLIFIED DEVICE FOR NUCLEIC ACID AMPLIFICATION ADN METHOD FOR
USING SAME
Abstract
The present invention relates to a disposable device (100) for
amplifying at least one target nucleic acid present in a liquid and
biological sample of interest, which consists of a solid body (2),
at least one fluid channel (3) connecting an inlet (4), via which
all or part of the sample of interest can be drawn up and/or
discharged, and an outlet (5), which is itself connected to a means
for the drawing up/discharging of the said sample of interest, the
fluid channel (3) further comprising from the inlet (4) to the
outlet (5): a first compartment (8) containing all or part of the
thermostable constituents, a means (15) for mixing the constituents
with the sample of interest, a second compartment (9) containing
all or part of the non-thermostable constituents, and in addition,
at least one zone intended for heating the said sample of interest
(6) mixed with the said amplification constituents in order to
allow the amplification of the target nucleic acid. The invention
also proposes an amplification method using such a device. The said
invention has a preferred application in the field of medical
diagnosis.
Inventors: |
Broyer; Patrick; (St
Cassien, FR) ; Drazek; Laurent; (Grenoble, FR)
; Dupont Filliard; Agnes; (Les Adrets, FR) ; Guy;
Michel; (Grenoble, FR) ; Pinston; Frederic;
(Grenoble, FR) ; Ponsard-Fillette; Magaly;
(Grenoble, FR) ; Kollaroczy; Thierry; (Engins,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
bioMerieux, S.A. |
Marcy L'Etoile |
|
FR |
|
|
Family ID: |
42135587 |
Appl. No.: |
15/643577 |
Filed: |
July 7, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13395348 |
Mar 9, 2012 |
9707554 |
|
|
PCT/FR2010/051936 |
Sep 17, 2010 |
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15643577 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/0217 20130101;
B01L 7/52 20130101; B01L 2400/086 20130101; B01L 2200/16 20130101;
B01L 2400/0478 20130101; B01L 2200/0621 20130101; B01L 3/5027
20130101; B01L 2300/0864 20130101 |
International
Class: |
B01L 3/02 20060101
B01L003/02; B01L 3/00 20060101 B01L003/00; B01L 7/00 20060101
B01L007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 18, 2009 |
FR |
0904469 |
Claims
1. Disposable device (1) for amplifying at least one target nucleic
acid present in a liquid and biological sample of interest (6),
which consists of a solid body (2), at least one fluid channel (3)
connecting an inlet (4), via which all or part of the sample of
interest (6) can be drawn up and/or discharged, and an outlet (5),
which is itself connected to a means (7) for the drawing
up/discharging of the said sample of interest, the fluid channel
(3) further comprising from the inlet (4) to the outlet (5): a
first compartment (8) containing all or part of the thermostable
constituents (12) required for producing the amplification, a means
(15) for mixing the constituents (12 optionally combined with 13)
with the sample of interest (6), a second compartment (9)
containing all or part of the non-thermostable constituents (13)
required for producing the amplification, and in addition, at least
one zone intended for heating (11) the said sample of interest (6)
mixed with the said amplification constituents (12 combined with
13), in order to allow the amplification of the target nucleic
acid.
2. Device according to claim 1, for detecting amplicons,
characterised in that it further comprises, in the second
compartment (9), all or part of the detection constituents required
for detecting the amplicons.
3. Device according to claim 1, for detecting amplicons,
characterised in that it further comprises, in the fluid channel
(3), a third compartment (10) containing all or part of the
constituents required for detecting the amplicons.
4. Device according to claim 3, characterised in that the third
compartment (10) is located between the second compartment (9) and
the outlet (5) of the device (1).
5. Device according to any one of claims 1 to 4, characterised in
that the inlet (3) accommodates a cone (28) of a pipette (16) or
the tip (3) of the pipette has a pipette-cone-shaped
configuration.
6. Device according to any one of claims 1 to 5, characterised in
that the drawing up/discharging device (7) is of the piston type
such as, for example, a pipette (7).
7. Device according to any one of claims 1 to 6, characterised in
that the cross-section of the channel (3) is constant and that the
compartments (8, 9 and optionally 10) have a larger
cross-section.
8. Device according to any one of claims 1 to 7, characterised in
that the inlet (4) communicates with at least two fluid channels
(3).
9. Device according to any one of claims 1 to 8, characterised in
that the outlet (5) comprises at least two fluid channels (3).
10. Device according to any one of claims 1 to 9, characterised in
that the constituents (12 & 13) are formed of freeze-dried or
dried biological compounds, soluble in the sample of interest
(6).
11. Device according to any one of claims 1 to 10, characterised in
that the drawing up/discharging means (7) is an integral part of
the disposable device (1).
12. Device according to claim 11, characterised in that the drawing
up/discharging means (7) comprises a cylinder (17) connected to the
fluid channel (3) and a piston (18) moving within the cylinder (17)
manually or by means of an actuator.
13. Device according to any one of claims 1 to 12, characterised in
that the mixing means (15) consists of the fluid channel (3), the
routing of which comprises at least one baffle (19).
14. A method for amplifying at least one target nucleic acid,
present in a liquid and biological sample of interest, within a
disposable device which comprises a solid body, at least one fluid
channel connecting an inlet, via which all or part of the sample of
interest can be drawn up and/or discharged, and an outlet, which is
itself connected to a device for the drawing up and/or discharging
of the sample of interest, the fluid channel further comprising
from the inlet to the outlet: a first compartment containing all or
part of thermostable constituents required for producing the
amplification, a means for mixing the thermostable constituents,
optionally combined with non-thermostable constituents, with the
sample of interest, a second compartment containing all or part of
the non-thermostable constituents required for producing the
amplification, and at least one zone intended for heating the
sample of interest mixed with the thermostable and non-thermostable
amplification constituents, in order to allow the amplification of
the target nucleic acid, which method consists in: (a) drawing up
via the inlet all or part of the sample of interest within the
device, (b) moving the said sample to the first compartment for
dissolving the thermostable amplification constituents therein, (c)
mixing the sample and the thermostable constituents with the mixing
means, (d) applying a first temperature gradient in order to
denature the nucleic acid of interest, (e) moving the mixture to
the second compartment for dissolving the non-thermostable
amplification constituents therein, (f) mixing the mixture and
non-thermostable constituents with the mixing means, and (g)
applying at least one second temperature gradient in order to
amplify the denatured nucleic acid.
15. The method according to claim 14, characterised in that the
thermostable amplification constituents of step (b) also contain
restriction enzymes, which are not necessarily thermostable, but
which allow the digestion of the nucleic acids of interest, which
are deoxyribonucleic acids (DNA), prior to the application of the
first temperature gradient of step (d).
16. Method according to either of claims 14 and 15, characterised
in that, after step (g), it consists in: (h) mixing mixture
(6+12+13), (i) moving the new mixture (6+12+13) to dissolve the
detection constituents (14) therein, and (j) detecting the presence
of amplicons.
17. The method according to claim 14, characterised in that the
amplification is a PCR amplification, for which the first
temperature gradient is between 90 and 100.degree. C., and the
second temperature gradients are an alternation of the temperature
in three different steps: between 90 and 100.degree. C. for the
first denaturation temperature, preferably about 94.degree. C.,
between 50 and 60.degree. C. for the second hybridisation
temperature, preferably about 55.degree. C., between 70 and
75.degree. C. for the third polymerisation temperature, preferably
about 72.degree. C.
18. The method according to claim 14, characterised in that the
amplification is a post-transcriptional amplification (NASBA or
TMA), for which the first temperature gradient is between 60 and
70.degree. C., preferably about 65.degree. C., and the second
temperature gradient is between 40 and 50.degree. C. for the second
polymerisation temperature gradient.
19. The method according to claim 21, characterised in that the
first temperature gradient is applied to the first compartment
and/or to the mixing means and that the second temperature
gradient(s) is/are applied to the mixing means and/or to the second
compartment and/or to the third compartment.
20. The method according to claim 14, characterised in that the
first temperature gradient is applied for 5 to 20 minutes, and that
the second temperature gradient(s) is/are applied: in the case of a
PCR amplification: for the denaturation, for less than one minute,
for the hybridisation, for less than one minute, and for the
polymerisation, for less than two minutes, in the case of a
post-transcriptional amplification, for less than two hours.
21. The method according to claim 14, characterized in that the
device further comprises, in the fluid channel, a third compartment
containing all or part of constituents required for detecting
amplicons.
22. The method according to claim 21, characterized in that the
cross section of the channel (3) is constant and that the third
compartment (10) has a larger cross-section.
23. The method according to claim 21, for detecting amplicons,
characterised in that, after step (g), it consists in: (h) moving
the mixture to the third compartment to dissolve the detection
constituents therein, (i) mixing the mixture, and (j) detecting the
presence of amplicons.
24. A method according to claim 14, characterised in that the
drawing up/discharging means comprises a cylinder connected to the
fluid channel and a piston moving within the cylinder manually or
by means of an actuator.
25. A method according to claim 14, characterised in that the
mixing means consists of the fluid channel, the routing of which
comprises at least one baffle.
26. The method of claim 20, wherein the first temperature gradient
is applied for 15 minutes.
27. The method of claim 20, wherein the second temperature gradient
applied in the case of a PCR amplification for the denaturation is
from 2 to 20 seconds.
28. The method of claim 20, wherein the second temperature gradient
applied in the case of a PCR amplification for the denaturation is
5 seconds.
29. The method of claim 20, wherein the second temperature gradient
applied in the case of a PCR amplification for the hybridisation is
from 2 to 20 seconds.
30. The method of claim 20, wherein the second temperature gradient
applied in the case of a PCR amplification for the hybridisation is
5 seconds.
31. The method of claim 20, wherein the second temperature gradient
applied in the case of a PCR amplification for the polymerisation
is from 5 to 80 seconds.
32. The method of claim 20, wherein the second temperature gradient
applied in the case of a PCR amplification for the polymerisation
is 10 seconds.
33. The method of claim 20, wherein the second temperature gradient
applied in the case of a post-transcriptional amplification is from
5 to 80 minutes.
34. The method of claim 20, wherein the second temperature gradient
applied in the case of a post-transcriptional amplification is
about 60 minutes in the case of RNA target nucleic acids or about
90 minutes in the case of DNA target nucleic acids.
Description
[0001] The present invention relates to a disposable device for
amplifying at least one target nucleic acid. The present invention
also relates to a method for amplifying such a target nucleic acid,
using an abovementioned device. The device, like the method, can be
used with any type of amplification technique, such as PCR, or a
post-transcriptional amplification technique, such as TMA or
NASBA.
[0002] The prior art is represented by document WO-A-99/33559 which
relates to a built-in reaction cartridge for handling fluids, but
also to document WO-A-2006/132886, from the same applicant, which
relates to a method and an apparatus for storing and using reagent
beads. This system comprises a compact instrument having four
identical and demountable modules, and a cartridge having a
plurality of wells, which incorporates a multichannel valve. A
piston serves to move the liquids from one well to another of the
cartridge in order to mix them. Among the various zones of the
cartridge, each is suitable either for: [0003] filtration on silica
matrix to obtain a capture, a purification and a concentration of
the sample analysed, [0004] lysis which, by means of glass beads
and under the action of ultrasound generated by an instrument,
serves to lyse the cell membranes of the cells present in the
sample, [0005] amplification which is carried out by transfer of
all or part of the eluate into the amplification and detection zone
(PCR) of the cartridge. The cartridge may include solid or liquid
reagents or both simultaneously.
[0006] This system is therefore fully built in and automated, with
a fairly short result return time (about one hour).
However, owing to the concept selected, the cartridge is relatively
complex and costly. It is therefore unsuitable for a
high-sample-rate system. Moreover, it is not suitable for
performing several tests per sample except by multiplexing the
amplification primers and/or detection probes in the same volume.
This makes the development of biological tests much more complex
and time-consuming, with inevitable compromises in terms of
detection performance.
[0007] Our invention is far more suitable for high rates because
the card that we protect can be easily handled by a robot, like a
simple pipetting cone.
Moreover, its lateral size is reduced, allowing the feasibility of
a high rate architecture using a fluorescence reading carousel
having an acceptable diameter for a system designed for a high
sample rate. Finally, the elongated design, with a plane surface,
makes it easy to move the card within a reading carousel which
requires: [0008] ensuring good thermal contact for the thermal
cycles (PCR type) or a constant and uniform temperature (NASBA, TMA
type) while allowing the movement of the said card by sliding on
the incubation blocks, and [0009] making a fluorescent reading, the
card being easily movable in front of the various read heads
(various wavelengths). High sample rate means a rate higher than
300 tests per day, with a low cost per test and reduced size.
[0010] In comparison with this system, the present invention helps
to obtain costs per test that are compatible with routine viral or
microbiological diagnosis, at high sample rate, but also for
portable tests for patients, also called POCT (Point of Care
Testing), thanks in particular to the simplicity of the consumable
developed, to its built-in pipetting function, and to the smaller
amplification reaction volume, which helps reduce the cost per test
(enzymes in molecular biology constitute the main portion of this
cost per test for the "reagents" part and a decrease in reaction
volume therefore helps to obtain a reduction proportional to the
reduction in volume) (5 .mu.L instead of 50 to 80 .mu.L in these
applications).
[0011] The prior art also includes document WO-A-2004/004904 which
relates to an apparatus designed to perform rapid self-contained
and mobile tests, in the particular context of bio-terrorism,
biological warfare and POCT, the apparatus requiring a minimum of
manual operations. This system uses a cartridge provided with inlet
ports connected to a flexible bag having one or more compartments
in which the amplification reagents (PCR) are freeze-dried. The
fluorescence is read directly through the deformable flexible film.
The bags are kept under vacuum, thereby making it possible, after
the introduction of the nucleic acid samples via the consumable
inlet port, to carry out an automatic and calibrated filling and to
dissolve the freeze-dried reagents prior to the PCR
amplification.
[0012] This device is unsuitable for performing routine tests at a
high sample rate. This vacuum fluid distribution and/or division
system is effective for one-step fluid protocols (filling) usable
in the case of a PCR amplification. However, it is unsuitable for
the method for amplifying nucleic acids, which require placing a
plurality of reagents in suspension consecutively, as in the case
for NASBA or TMA amplifications. This therefore requires adding a
valve and maintaining a partial vacuum between the two chambers
containing the amplification reagents, causing a complexification
of the consumable and of the associated instrument. Moreover, the
consumable is not suitable for directly drawing up the sample
containing the nucleic acids (deposition by pipette) without manual
action; hence there is no pipetting function. Furthermore, this
device, which combines a rigid portion with a flexible bag portion,
is not easily manageable in an architecture in which the user can
place a sample at any time, even during the operation of the
instrument, called a Random Access architecture.
[0013] Our invention, on the contrary, proposes a card which can
easily be handled by a robot and can be used as a cone to pipette
solutions, mix them, take up the eluate and draw it up to conduct
reactions such as amplification reactions. The card, according to
the present invention, can therefore be used as a pipette cone for
making additions, drawing up liquid reagents into tubes, and
thereby carrying out the steps prior to the amplification/detection
step. With suitable automation, the said card can also sample and
withdraw a plurality of cones simultaneously. For this purpose,
embodiments comprising more than one fluid channel are presented in
the rest of this document.
[0014] The present invention of the Applicant, by its original
design, serves to adapt both to the PCR (or RT-PCR) amplification
protocol only requiring one reagent (and therefore feasible in a
single chamber) and the two-step amplification protocol requiring
separation of the amplification reagents in two distinct chambers,
as in the case of post-transcriptional amplifications. The
invention also solves the problem of instrument architecture, by
proposing a consumable (or card) usable either in the POCT system
(a few tests per day) or in a routine high-rate diagnosis system
(more than 300 tests per day) by integrating the pipetting
function, which uses conventional cones (added on or built in).
[0015] The prior art also includes document WO-A-2007/100500. This
concerns a highly oriented POCT system or the low rate in molecular
biology. This easy-to-use system makes it possible to work directly
from a drop of blood (a few tens of .mu.L). The associated
instrument is also compact thanks to a combination of actuators
designed to isolate the compartments of the tubular consumable and
thereby allow the transfer and mixing of the liquids in the order
defined in manufacture by the prior filling of the consumable.
[0016] A major drawback of this system is its lack of flexibility
for the biological protocol to be followed. On the one hand, the
sample and buffer volumes (magnetic nucleic acid capture particles,
washing and elution buffers) are fixed by the volume of each
compartment of the consumable, as defined during the manufacture of
the consumable. It is known that it is often necessary to modify
the biological protocol, in order to obtain better nucleic acid
capture, amplification and detection performance according to
various parameters, such as, for example, the type of biological
sample treated, the presence or absence of inhibitors (spit, LBA,
plasma, urine, whole blood, etc.). With this prior art system,
modifying the protocols requires creating a different consumable
every time with different volumes for each compartment, and thereby
modifying the sealing zones, with a very strong limitation
associated with the location of the zones where the mobile
actuators are installed in the associated instrument (valves and
pistons for the rupture of the said sealing zones by overpressure).
Furthermore, the sample volume remains very small and therefore
does not cover all the needs of the users, particularly in tests
with several milliliters of sample. Moreover, the flexible object
is difficult to manage by a robot except at high extra cost. It has
no pipetting function, nor flexibility in the choice of the elution
and washing volumes, and therefore has less flexibility in sample
preparation.
[0017] The applicant has also filed a number of documents which can
constitute the prior art. These concern in particular patent
application EP-B-1.187.678 which relates to a device for using an
analysis card in which fluid reaction and transfer steps are
carried out under the action of control means built into the
card.
[0018] One problem of this type of device is that it is forced to
operate with the help of valves which consist of elements that are
deformable under the action of an actuator, thereby causing the
direct or indirect closure of the channels associated with the
valves. The essential problem with this device is its complexity.
Thus, the presence of valves considerably complicates the
manufacture of the analysis card thus formed and adds to its
production cost, and furthermore, many external elements
(actuators) are needed to actuate all the said valves.
[0019] The present invention proposes to solve the problems
highlighted by all the abovementioned prior art documents. For this
purpose, it proposes a device which is disposable and which
satisfies a number of technical characteristics.
[0020] In a particularly advantageous embodiment of the invention,
the device is a consumable which is considered like a pipette,
carrying the dried or freeze-dried reagents required for an
amplification of RNA or DNA targets from a reduced volume of
nucleic acids (5 to 10 .mu.L), thereby cutting the costs linked to
the reagents, and incorporating a valve to eliminate any risk of
contamination. This is a slide valve, normally open and then closed
after locating the reaction volume in the reading zone, when there
are no further steps to be carried out. This type of device has
many features unknown in the prior art: [0021] Based on this
concept, the ability to manufacture robots in a POCT version
(processing by batch of 8-24 samples per batch) and in a high-rate
robot version using the same consumable. [0022] Automatic sampling,
by moving the tip that is added on or is an integral part of the
inventive device, of the volume of purified nucleic acid serving to
reduce the number of manual steps, thereby consequently serving to
simplify the automation of a robot apparatus using such
consumables. [0023] Ability to incorporate, in a single associated
instrument, a PCR or NASBA amplification for POCT. [0024]
Simplification of the instrumentation by the inclusion in the
device of carried and freeze-dried or dried reagents, which can be
dissolved and mixed consecutively by simple movement within the
said device, the configuration of the fluid circuit of which
facilitates this dissolution and this mixing. [0025] Ability to
perform mono-tests (one set of amplification primers per fluid
channel within the device), multiplex tests (with at least two sets
of primers per channel) or by panel (at least two separate sets of
primers in at least two channels) from a common instrumental
architecture. [0026] Total automation of the amplification protocol
with an inexpensive device and compact associated instrumentation.
[0027] Shorter total amplification time (especially with NASBA) by
reducing the denaturation time in comparison with a "conventional"
amplification (1 minute versus 5 to 10 minutes) due to the heating
through the thin cover film of the reaction channel, instead of a
thicker plastic tube, whose thermal inertia is unfavourable. [0028]
No lifetime limitation of the enzyme compared to conventional
automation, because the reagents, carried in the said device,
remain dry until being taken up by the liquid sample to be
amplified.
[0029] The present invention relates to a disposable device for
amplifying at least one target nucleic acid present in a liquid and
biological sample of interest, which consists of a solid body, at
least one fluid channel connecting an inlet, via which all or part
of the sample of interest can be drawn up and/or discharged, and an
outlet, which is itself connected to a means for the drawing
up/discharging of the said sample of interest, the fluid channel
further comprising from the inlet to the outlet: [0030] a first
compartment containing all or part of the thermostable constituents
required for producing the amplification, [0031] a means for mixing
the constituents with the sample of interest, [0032] a second
compartment containing all or part of the non-thermostable
constituents required for producing the amplification, [0033] and
in addition, at least one zone intended for heating the said sample
of interest mixed with the said amplification constituents, in
order to allow the amplification of the target nucleic acid.
[0034] According to an embodiment, the device for detecting
amplicons is characterised in that it further comprises, in the
second compartment, all or part of the detection constituents
required for detecting the amplicons.
[0035] According to another embodiment, the device for detecting
amplicons is characterised in that it further comprises, in the
fluid channel, a third compartment containing all or part of the
constituents required for detecting the amplicons.
[0036] Also in another embodiment, the device further comprises, in
the fluid channel, a third compartment containing nothing but
serving for the subsequent detection of the amplicons in a clean
environment.
[0037] In an alternative embodiment of the device, described in the
previous paragraph, the third compartment is located between the
second compartment and the outlet of the device.
[0038] Regardless of the alternative embodiment, the inlet of the
device accommodates a cone of a pipette or the tip of the pipette
has a pipette-cone-shaped configuration.
[0039] Regardless of the preceding alternative embodiment, the
drawing up/discharging device is of the piston type such as, for
example, a pipette.
[0040] Regardless of the preceding alternative embodiment, the
cross-section of the channel is constant and the compartments have
a larger cross-section.
[0041] According to a multichannel embodiment, the inlet
communicates with at least two fluid channels.
[0042] Also according to a multichannel embodiment, the outlet
comprises at least two fluid channels.
[0043] Regardless of the preceding alternative embodiment, the
constituents are formed of freeze-dried or dried biological
compounds, soluble in the sample of interest.
[0044] Regardless of the preceding alternative embodiment, the
drawing up/discharging means is an integral part of the disposable
device.
[0045] According to the latter alternative embodiment, the drawing
up/discharging means comprises a cylinder connected to the fluid
channel and a piston moving within the cylinder manually or by
means of an actuator.
[0046] Regardless of the preceding alternative embodiment, the
mixing means consists of the fluid channel, the routing of which
comprises at least one baffle.
[0047] The present invention also proposes a method for amplifying
at least one target nucleic acid, present in a liquid and
biological sample of interest, made within a device previously
described, which consists in: [0048] (a) drawing up via the inlet
all or part of the sample of interest within the device, [0049] (b)
moving the said sample for dissolving the thermostable
amplification constituents therein, [0050] (c) mixing the sample
and the thermostable constituents, [0051] (d) applying a first
temperature gradient in order to denature the nucleic acid of
interest, [0052] (e) moving the mixture for dissolving the
non-thermostable amplification constituents therein, [0053] (f)
mixing mixture and non-thermostable constituents, and [0054] (g)
applying at least one second temperature gradient in order to
amplify the denatured nucleic acid.
[0055] According to an embodiment, the thermostable amplification
constituents of step (b) also contain restriction enzymes, which
are not necessarily thermostable, but which allow the cleavage,
into predetermined positions, of the nucleic acids of interest,
which are deoxyribonucleic acids, prior to the application of the
first temperature gradient of step (d).
[0056] According to another embodiment, which can be used in
addition to the abovementioned embodiment, the detection of the
amplicons consists, after step (g) in:
[0057] (h) moving the new mixture to dissolve the detection
constituents therein, [0058] (i) mixing mixture and detection
constituents, and [0059] (j) detecting the presence of
amplicons.
[0060] According to another embodiment, which can be used in
addition to at least one of the abovementioned embodiments, the
amplification is a PCR amplification, for which the first
temperature gradient is between 90 and 100.degree. C., and the
second temperature gradients are an alternation of the temperature
in three different steps: [0061] between 90 and 100.degree. C. for
the first denaturation temperature, preferably about 94.degree. C.,
[0062] between 50 and 60.degree. C. for the second hybridisation
temperature, preferably about 55.degree. C., [0063] between 70 and
75.degree. C. for the third polymerisation temperature, preferably
about 72.degree. C.
[0064] According to another embodiment, which can be used in
addition to at least one of the abovementioned embodiments, the
amplification is a post-transcriptional amplification (NASBA or
TMA), for which the first temperature gradient is between 60 and
70.degree. C., preferably about 65.degree. C., and the second
temperature gradient is between 40 and 50.degree. C. for the second
polymerisation temperature gradient.
[0065] According to another embodiment, which can be used in
addition to at least one of the abovementioned embodiments, the
first temperature gradient is applied to the first compartment
and/or to the mixing means and the second temperature gradient(s)
is/are applied to the mixing means and/or to the second compartment
and/or to the third compartment.
[0066] According to another embodiment, which can be used in
addition to at least one of the abovementioned embodiments, the
first temperature gradient is applied for 5 to 20 minutes,
preferably 15 minutes, and the second temperature gradient(s)
is/are applied: [0067] in the case of a PCR amplification: [0068]
for the denaturation, for less than one minute, preferably from 2
to 20 seconds, preferably 5 seconds, [0069] for the hybridisation,
for less than one minute, preferably from 2 to 20 seconds,
preferably 5 seconds, and [0070] for the polymerisation, for less
than two minutes, preferably from 5 to 80 seconds, preferably 10
seconds, [0071] in the case of a post-transcriptional
amplification, for less than two hours, preferably from 5 to 80
minutes, and even more preferably: [0072] about 60 minutes in the
case of RNA target nucleic acids, or [0073] about 90 minutes in the
case of DNA target nucleic acids.
[0074] The following terms can be used equally in the singular or
the plural.
[0075] The term "constituent" also means "reagent", "amplification
reagent", "extraction reagent", or "purification reagent" or "raw
material" which designate reagents, such as reaction buffers,
enzymes, mono-, bi- or triphosphate nucleosides, but also solvents,
the salts required for carrying out a nucleic acid extraction,
purification or enzymatic amplification reaction.
[0076] In the context of the present invention, "container" or
"plastic container" means any receptacle such as tubes, pipette
cones or tips, whether made from plastic (for example the Eppendorf
type) or from glass or from all other materials.
[0077] In the context of the present invention, "nucleic acid"
means a chain of at least two nucleotides, preferably at least ten
nucleotides selected from the four types of nucleotides of the
genetic code, that is to say, if the nucleic acid is a DNA: [0078]
dAMP (deoxyadenosine 5'-monophosphate), [0079] dGMP (deoxyguanosine
5'-monophosphate), [0080] dTMP (deoxythymidine 5'-monophosphate),
and [0081] dCMP (deoxycytidine 5'-monophosphate),
[0082] if the nucleic acid is an RNA: [0083] AMP (adenosine
5'-monophosphate), [0084] GMP (guanosine 5'-monophosphate), [0085]
UMP (uridine 5'-monophosphate), and [0086] CMP (cytidine
5'-monophosphate).
[0087] The nucleic acid may also optionally comprise at least one
inosine and/or at least one modified nucleotide. In the context of
the present invention, the term "modified nucleotide" means a
nucleotide, for example at least one nucleotide comprising a
modified nucleic base, deoxyuridine, diamino-2,6-purine,
bromo-5-deoxyuridine, or any other modified base, preferably with
the exception of 5-methyl-cytosine. The nucleic acid may also be
modified at the internucleotide bond, such as, for example,
phosphorothioates, H-phosphonates, alkyl-phosphonates, in the
structure such as, for example, alpha-oligonucleotides
(FR-A-2.607.507) or polyamide nucleic acids (PMA) (Egholm M. et
al.; J. Am. Chem. Soc.; 1992; 114; 1895-97) or
2'-O-alkyl-ribonucleotides and/or a 2'-O-fluoro nucleotide and/or
2'-amine nucleotide and/or an arabinose nucleotide, and LNA (Sun B.
W. et al., Biochemistry; 2004; Apr. 13; 43; (14): 4160-69). Among
the 2'-O-alkyl-ribonucleotides, the 2'-O-methyl-ribonucleotides are
preferred, but use can also be made of 5-Propinyl Pyrimidine
Oligonucleotides (Seitz O., Angewandte Chemie International Edition
1999; 38(23); Dec: 3466-69).
[0088] The term "nucleotide" defines either a ribonucleotide or a
deoxyribonucleotide.
[0089] In the context of the present invention, "biological sample"
or "liquid biological sample" means any sample that may contain
nucleic acids. The latter may be extracted from tissues, blood,
serum, saliva, circulating cells of a patient, or may originate
from a food, an agrifood, or may even be of environmental origin.
Extraction is carried out by any protocol known to a person skilled
in the art, for example by the isolation method described in patent
EP-B-0.369.063.
[0090] In the context of the present invention, "contaminant" or
"contaminant acid" or "contaminant nucleic acid" or "contaminant
element" means any nucleic acid whose amplification is not desired
and which is liable to generate a false positive result during the
detection.
[0091] "Amplification" or "amplification reaction" means any
nucleic acid amplification technique well known to a person skilled
in the art, such as: [0092] PCR (Polymerase Chain Reaction),
described in patents U.S. Pat. No. 4,683,195, U.S. Pat. No.
4,683,202 and U.S. Pat. No. 4,800,159, and its derivative RT-PCR
(Reverse Transcription PCR), in particular in a one-step format, as
described in patent EP-B-0.569.272. Preferably, the PCR is carried
out on a single strand with a single primer pair. [0093] LCR
(Ligase Chain Reaction), described for example in patent
application EP-B-0.201.184, [0094] RCR (Repair Chain Reaction),
described in patent application WO-A-90/01069, [0095] 3SR (Self
Sustained Sequence Replication) with patent application
WO-A-90/06995, [0096] NASBA (Nucleic Acid Sequence-Based
Amplification) with patent application WO-A-91/02818, [0097] TMA
(Transcription Mediated Amplification) with U.S. Pat. No.
5,399,491, and [0098] RCA (Rolling Circle Amplification) described
in U.S. Pat. No. 6,576,448.
[0099] In the context of the present invention, "target" or "target
nucleic acid" or "nucleic target" or "target of interest" or
"nucleic acid of interest" means a nucleic acid (an
oligonucleotide, a polynucleotide, a fragment of nucleic acid, a
ribosomic RNA, a messenger RNA, a transfer RNA) to be amplified
and/or detected. The target may be extracted from a cell or
chemically synthesised. The target may be free in solution or may
be bonded to a solid support.
[0100] The term "liquid and biological sample of interest" means a
homogeneous or heterogeneous aqueous solution.
[0101] "Solid support" means particles which may be made from
latex, glass (CPG), silica, polystyrene, agarose, sepharose, nylon,
etc. These materials may optionally allow the confinement of
magnetic material. They may also be a filter, a film, a membrane or
a strip. These materials are well known to a person skilled in the
art.
[0102] The target may be a viral, bacterial, fungal nucleic acid,
or yeast, present in a mixture, in the form of a single or double
strand of DNA and/or of RNA. In general, the target has a length of
between 50 and 10 000 nucleotides, but it is usually between 100
and 1000 nucleotides.
[0103] "Marker" means a molecule carried by a nucleotide. The link
between the marker and the nucleotide can be made in various ways
known to a person skilled in the art. Manual coupling is carried
out by using markers carrying an activated group, typically a
carboxyl or a thiol, which are coupled onto a modified internal
nucleotide carrying the corresponding reagent group (amine or
thiol, for example), or on one end of the modified nucleotide
strand with these same reagent groups. Automatic coupling is
obtained by using phosphoramidites carrying the marker, and the
coupling is then carried out during the automated synthesis of the
nucleotide strand, either on one end of the strand, or on an
internal position, according to the type of phosphoramidite used.
The marker may be a fluorophore or a fluorescence quencher.
[0104] "Fluorophore" means a molecule which emits a fluorescence
signal when excited by light at a suitable wavelength. The
fluorophore may in particular be a rhodamine or a derivative
thereof such as Texas Red, a fluorescein or a derivative thereof
(for example FAM), a fluorophore of the Alexa family such as Alexa
532 and Alexa 647, Alexa 405, Alexa 700, Alexa 680, Cy5 or any
other fluorophore appropriate to the measuring instrument employed.
Fluorophores available for detection probes are widely varied and
known to a person skilled in the art.
[0105] In the context of the present invention, "fluorescein" means
an aromatic chemical molecule which emits a fluorescence signal
with an emission peak around 530 nm, when excited by light at a
wavelength of about 490 to 500 nm, preferably 495 nm.
[0106] "Fluorescence quencher" or "quencher" means a molecule which
interferes with the fluorescence emitted by a fluorophore. This
quencher may be selected from non-fluorescent aromatic molecules,
to avoid interfering emissions. Preferably, the said quencher is a
Dabsyl or a Dabcyl or a black hole Quencher.TM. (BHQ) which are
non-fluorescent aromatic molecules that prevent the emission of
fluorescence when they are in the physical vicinity of a
fluorophore. The fluorescence resonance energy transfer (FRET)
technique can also be used as described for example in Fluorescent
Energy Transfer Nucleic Acid Probes, p. 4, Ed. V. V. Didenko,
Humana Press 2006, ISSN 1064-3745. The quencher may also be
selected from fluorescent molecules, such as for example TAMRA
(carboxytetramethylrhodamine).
[0107] The "three-base detection probe" or "three-base probe",
called S3B, is a probe as defined previously and which, in addition
to the preceding features, consists of a nucleotide chain of three
different types of bases selected from the group of adenine,
thymine, guanine, cytosine. It is well understood by a person
skilled in the art that according to the form of the probes
(Molecular Beacon, see EP95/904 104.7, EP96/303 544.9 and EP97/923
412.7, or O-probe, see PCT/FR2009/051315, etc.), the probe will
consist of a portion of a sequence wherein the nucleotide chain
comprises nucleotides of four different types of bases and a
sequence wherein the nucleotide chain comprises nucleotides of
three different types of bases (sequence allowing the hybridisation
and detection of the amplicons).
[0108] In certain cases, to improve the hybridisation with the
amplicons and hence their detection, the probes according to the
invention may occasionally contain uracile instead of thymine. In
this case, the probes according to the invention will consist of a
nucleotide chain of four different types of bases (uracile,
guanine, adenine, thymine).
[0109] "Hybridisation" means the process during which, under
suitable conditions, two single-strand nucleotide fragments, having
complementary sequences in whole or in part, are capable of forming
a double strand or "duplex" stabilised by hydrogen bonds between
the nucleic bases. The hybridisation conditions are determined by
the stringency, that is to say, the rigour and the low salinity of
the operating conditions. Hybridisation is increasingly specific
when carried out at higher stringency. Stringency is defined in
particular according to the composition in bases of a probe/target
duplex, and also by the degree of mismatch between two nucleic
acids. The stringency may also be a function of the reaction
parameters, such as the concentration and type of ionic species
present in the hybridisation solution, the type and concentration
of denaturing agents and/or the hybridisation temperature. The
stringency of the conditions under which a hybridisation reaction
must be conducted will mainly depend on the hybridisation probes
employed. All these data are well known and the appropriate
conditions can be determined by a person skilled in the art.
[0110] The appended examples and figures represent particular
embodiments and cannot be considered as limiting the scope of the
present invention.
[0111] FIG. 1 shows a first embodiment of the inventive disposable
device, which contains a single fluid channel. In this particular
configuration, the device is ready to draw up a biological sample
to be treated in which at least one target nucleic acid is likely
to be present.
[0112] FIG. 2 also shows this first embodiment, but in another
particular configuration, in which the device is in the course of
drawing up the biological sample to be treated (not shown in this
figure).
[0113] FIG. 3 shows a second embodiment of the inventive disposable
device, which contains two fluid channels in parallel. In this
particular configuration, the device is ready to draw up the said
biological sample containing at least one target nucleic acid,
likely to be present.
[0114] FIG. 4 also shows this second embodiment, but in another
particular configuration, in which the device is in the course of
drawing up the biological sample to be treated (not shown in this
figure).
[0115] FIG. 5 shows a third embodiment of the inventive disposable
device, which contains eight fluid channels in parallel. In this
particular configuration, the device is ready to draw up the said
biological sample containing at least one target nucleic acid,
likely to be present.
[0116] FIG. 6 shows a cross-section along A-A of FIG. 5 for better
visualising the way in which the drawing up and discharging actions
are conducted within the said device.
[0117] Finally, FIGS. 7 to 10 show the device according to an
alternative of the second embodiment, but in the course of use:
[0118] FIG. 7: The disposable device does not contain the liquid
sample of interest. Its tip is just in contact with the sample
which is present in a container.
[0119] FIG. 8: The tip being in contact with the sample, the
pistons are moved for drawing up the sample of interest into the
disposable device. In this case, the sample is located in the first
compartment and the thermostable constituents which it contains.
Before the pistons have completed their drawing up in this phase,
the disposable device is raised and/or the container is lowered, so
that the device and sample of the said container are no longer in
contact and the single channel is only filled with air, as is the
case in this FIG. 8. The two downstream channels each contain a
liquid column of an aliquot of the sample. Preferably, the drawing
up by the pistons is stopped when the device is withdrawn from the
sample remaining present in the container.
[0120] FIG. 9: The drawing up of the sample of interest by the said
pistons continues and the said sample is transferred to the mixing
means, where the back-and-forth motions allow the proper mixing of
the sample and the thermostable constituents. The combination of
the sample with the thermostable constituents will continue to be
called "sample" for easier reading and understanding.
[0121] FIG. 10: The pistons are moved to allow the drawing up of
the sample of interest into the disposable device. In this case,
the sample is located in the second compartment and the
non-thermostable constituents that it contains.
[0122] FIG. 11: The drawing up is suspended, but the sample of
interest is discharged by the said pistons and the said sample is
transferred to the mixing means, where the back-and-forth motions
allow the proper mixing of the sample and the non-thermostable
constituents. Once again, the combination of the sample with the
thermostable and non-thermostable constituents will continue to be
called "sample" for easier reading and understanding.
[0123] FIG. 12: The pistons are moved to allow the drawing up of
the sample of interest into the disposable device. In this case,
the sample is located in the third compartment and the constituents
necessary for detection that it contains.
[0124] FIG. 13 shows NASBA amplification curves for the prior art
(EasyQ) for increasing values of the quantity of target (see scale
from 0 to 10 000 in the high position--see reference to the arrow
B). The curves corresponding to the amplification of the HIV target
sequence are plotted in the lower position (see reference to the
arrow A), and the curves corresponding to the calibrator are in the
upper position.
[0125] FIG. 14 shows the NASBA amplification curves for the
invention under the same conditions as those applied in FIG. 13
(similarly for the use of the arrows A and B). It should be noted
that to improve the legibility of this figure, the curves
corresponding to the HIV target have been expanded by a factor of
5. Thus, with the device of the present invention, the curves
corresponding to the target (HIV) vary from about 8 to 40 rfu
(Relative Fluorescence Units). Since the curves of the calibrator
vary from 50 to 250, the graph is not legible if the two curves are
plotted with the same scale. As plotted, the HIV curves vary
between 40 and 200 and are therefore more legible. Finally, these
are relative values without prejudice to the effectiveness of the
amplification, and it is actually the inflection points of each
curve which have interpretative value.
[0126] FIG. 15 proposes the variable quantification calculated by
the algorithm HIV v.2.0 for the EasyQ instruments (left) and
according to the invention (right).
[0127] FIG. 16 shows the quantification result as a function of the
quantity of target.
[0128] Finally, FIG. 17 shows a longitudinal section along B-B of
FIG. 2 of the inventive device.
[0129] The present invention is clearly represented in the set of
FIGS. 1 to 17. Three embodiments are shown more particularly.
[0130] A first embodiment shown in FIGS. 1 and 2 represents a
perfectly simple embodiment of the present invention. It consists
of a disposable device formed of a solid body 2 in the surface of
which a fluid circuit or channel 3 is etched. This fluid circuit 3
is obviously bounded by a film of the BOPP (Bi-Oriented
PolyPropylene) type, not referenced in these two figures, but
present with the reference 14 in FIG. 17, which prevents the liquid
sample from leaving the said circuit 3. The fluid channel 3
comprises, on the one hand, a through hole 4, called inlet, at the
bottom of each figure, and another through hole, called outlet, at
the top of each figure, which enables the channel 3 to have two
output openings. In fact, the inlet 4 is formed by a pipette tip,
referenced 16, that is to say, moulded in a single part with the
overall body 2. FIG. 1 shows an added on pipette tip 27, showing a
particular embodiment using a conventional tip. On the other side,
the outlet 5 is present in a cylinder 17, which constitutes one of
the parts of the drawing up/discharging means 7. The other part of
this drawing up/discharging means 7 consists of a piston 18 which
can slide within the cylinder 17, moved by an actuator, not shown
here, along the arrow F1, on the one hand, which is a movement of
the piston 18 in the cylinder 19 in which the volume of fluid in
the fluid circuit 3 decreases, or along arrow F2, which is a
movement of the piston 18 in the cylinder 19 which is completely
different from the previous one, that is to say, it increases the
volume of fluid in the fluid circuit 3. The actuator can act on the
piston via a linkage means 21. This system is particularly
advantageous in the case in which the overall system is to be
automated. In this embodiment in FIGS. 1 and 2, it should be
observed that the fluid circuit is relatively simple because along
the channel 3, having a substantially constant cross-section, it
comprises a first compartment 8, a second compartment 9 and,
between the compartments 8 and 9, a mixing means 15 consisting of a
set of baffles 19. The roles of these compartments 8 and 9 and of
this mixing means 15 are described more amply below.
[0131] The second embodiment is shown in FIGS. 3 and 4. This is an
embodiment that is substantially identical to the previous one, but
in which two fluid channels are in parallel with one another. This
device is referenced 100 although all the other elements have the
same reference numerals as those previously used. FIG. 3 is
substantially identical to FIG. 1 because the piston 18 is in the
rest position, that is to say, it is in the low position in the
cylinder 17, and the actuator or the manipulator moves the said
piston 18 via the linkage means 21. In this embodiment, there is
therefore an inlet 4, a single channel 3 on the downstream portion,
which is then split into two channels of identical cross-section
which rise in parallel along the body 2 of the device 100. Along
each of the channels 3, as in the previous case, are located a
first compartment 8, a mixing means 15 and a second compartment 9.
However, there is also a third compartment 10 upstream. For
practical reasons, a trap is formed between the second and third
compartments 9 and 10, that is to say, the first bend of the trap
is located above the first compartment 9 and the third compartment
10 is located at the second bend of the trap. Alternatively, the
compartment 10 can be deleted and the reading made directly in the
compartment 9. The role of the compartment 9 or 10 is primarily to
secure any undesired movement of the liquid towards the lower part
of the card, the said card being used in the vertical position, and
then, to increase the volume of liquid usable for the fluorescence
detection by maximising the diameter of the reading lid, made in
the associated heating unit in the instrument, and to position it
opposite the compartment 9 or 10. Another feature of this
embodiment is that for an inlet 4, there are two outlets 5, because
each channel 3 is connected upstream to a drawing up/discharging
means 7. Hence each channel 3 is associated with a cylinder 17 and
with a piston 18. In this particular embodiment, the two pistons 18
are independent of one another, as clearly shown in FIG. 4, that is
to say, they can be actuated along F1 and F2 independently from one
another.
[0132] The configuration with two individualised pistons, moving
independently of one another, serves to correct the positioning
defect of the liquid segments in each fluid channel individually,
in particular by a recalibration of the said segments in the
detection zone, for example, the shift possibly being due to an
inhomogeneous viscous sample, a slight moulding defect in the fluid
circuit of the card, or an undesired movement due to a thermal
gradient momentarily creating a pressure differential on either
side of the fluid segment. Hence this serves to increase the
operating robustness of the device. This system operates whenever
there is more than one channel, with the help of position sensors
placed at appropriate locations, on the one hand, within the
device, and on the other hand, with regard to the card according to
the invention.
[0133] It should be noted that it is particularly advantageous to
have an anti-extraction system for each piston 18. Thus, and
advantageously, each piston 18 can be provided with a guide 23 that
is connected by one end to the upper end of the piston rod 18 and
at its other end, not shown in the figures, to a larger-section
form preventing the accidental extraction of the post-amplification
piston during handling by the operator (e.g. unloading of the
consumable from the instrument at the end of analysis, or piston
positioning error by the instrument). This anti-extraction system
is obviously adaptable to all the embodiments considered by the
present invention.
[0134] FIGS. 5 and 6 show a third embodiment which is substantially
identical to the previous one, except in that it comprises eight
fluid channels 3 in parallel. As for the two preceding embodiments,
there is only one inlet 4 located at a tip 16 from which a single
channel leaves. This is divided into two to three repetitions,
yielding a total number of eight channels 3 in the active portion
of the device 200 and therefore eight outlets 5. Each channel 3
then consists of: [0135] a first compartment 8 followed by a mixing
means 15 composed of a number of baffles 19, [0136] a second
compartment 9, and finally [0137] a third compartment 10. Each
channel obviously terminates in a drawing up/discharging means 7
consisting, as usual, of a cylinder 17 and a piston 18. Similarly
to the second embodiment, this third embodiment comprises pistons
18 which are handleable via the linkage means 21 in a
self-contained and independent manner with regard to the other
adjacent pistons 18.
[0138] FIG. 6 shows a longitudinal section along the axis A-A of
FIG. 5, for better visualisation of the connection existing between
the channel 3 and the drawing up/discharging means 7. This section
serves to show the body 2 of the device 200, which comprises on its
upper surface a channel 3 bounded externally by a partitioning film
14 made from an appropriate material. The film is preferably made
from BOPP (Bi-oriented PolyPropylene) with a silicon cement, but
may also be made from PP (PolyPropylene), PET (PolyEthylene
Terephthalate), TPE (ThermoPlastic Elastomer), or PP/PE type
complex film which can be sealed by laser around the fluid
channels. This channel 3 culminates in a transverse hole 25 which
terminates in the cylinder 17. Present in this cylinder 17 is the
piston 18, the piston head 26 of which forms a seal with the sleeve
of the said cylinder 17. The piston head 26 may be composed of two
parts (body with groove and elastomer O-ring) or may consist of a
one-piece piston, simplifying the card assembly operations and
serving to reduce the cost per test. It is therefore obvious that
when the actuator or the manipulator applies a force along F2 on
piston 18, the latter allows the drawing up of the gaseous or
liquid fluid present in the channel 3, while the action along F1
serves to discharge this fluid via the outlet 4, not shown in FIG.
6.
[0139] Built-in pistons can be manufactured in two different ways.
One is a simple piston with or without ring segment ("O-"), as in
the above case, or a two-section piston. Although this type of
piston is well known to a person skilled in the art, the use of a
two-section piston has the following advantages: [0140] The
intrinsic positioning accuracy is improved compared to a simple
piston (having the same diameter) because the volume of liquid
pumped is determined/calibrated by the difference in volume between
the two sections of the said piston (the drawing up function is
therefore located between the two diameters of this piston). [0141]
After the pumping action, the piston is completely thrust into the
sleeve/cartridge in which it is moved. This eliminates any risk
that the piston will be extracted by user error.
[0142] FIGS. 7 to 14 show the second embodiment of FIGS. 3 and 4
during the use of the device 100. It should be noted that this
device 100 is very slightly different from the one already
described, because the two pistons in the present case are joined
together so that the fluid motion in each of the two channels 3 is
identical. This is obviously one alternative and the pistons can
also be detached from one another, either automatically by the
robot, or manually by the manipulator as required. It is also
possible that the bridge connecting the two pistons can be deformed
without necessarily being physically cut.
[0143] FIG. 7 shows that the liquid and biological sample 6 is
exclusively contained in a container 20 although the latter 6 is in
contact with the device 100; in particular, the tip 16 is partially
immersed in the said liquid 6. The device 100 comprises, in
addition to that which was shown in FIGS. 3 and 4, thermostable
constituents 12 present in the first compartment 8 and
non-thermostable constituents 13 in the second compartment 9. In
fact, the constituents are stored in solid form, for example
according to the technical data given in patent EP-B-0.641.389,
which the reader is requested to consult for further details on
this subject.
[0144] It is obviously conceivable for there to be only two
compartments 8 and 9, as in FIGS. 1 and 2. In this case, it is
necessary to have non-thermostable and detection constituents 13
and 14 which are present together in the second compartment 9.
Similarly, if an amplification only having thermostable
constituents is used, it is also possible to have all the
ingredients present in a single compartment, 8 or 9 in particular.
Preferably, the tip is matched to a pipetting cone 27, as shown in
FIG. 1, for example with a capacity of 50 to 200 .mu.L, or
otherwise, which is not shown in the figures but is well known to a
person skilled in the art, a polyethylene straw which can be
thermally sealed after all the fluid sequences are produced in the
card. In FIG. 7, either the container of the biological sample can
rise in contact with the tip, or the card is brought by the
instrument into contact with the liquid present in the container,
the latter case corresponding to architectures for high-rate
machines. The device will then be used as a conventional pipetting
cone and will therefore be moved along the axes X, Y and/or Z by a
robot to take a sample from the container or containers.
[0145] In FIG. 8, the linkage means 21 is moved along F2, so that
the pistons 18 draw up the liquid sample present in the container
20 within the card. At the end of this step, not shown in this
figure, the device 100 has been raised whereas the pistons 18
continue their drawing up action, explaining the presence of two
liquid columns in each of the channels 3. It should be noted that
the volume drawn up is related to the cross section of the piston
built into the card and to the length of movement of the piston.
Apart from the optional initial stop in the pipette cone, the first
stop of each liquid sample 6 occurs in the first compartment 8,
that is to say, at the thermostable constituents 12, thereby making
it possible to dilute the said constituents which are actually
stored in freeze-dried or dried form, as is the case for the other
constituents 9 and 10. The liquid segment is kept on the reagent
position to make it easier to place the dried or freeze-dried
reagent in suspension again, typically for ten seconds, generally
less than one minute.
[0146] In FIG. 9, the fluid continues to move along F4 towards the
mixing means 15. This movement along F4 corresponds to the drawing
up of the piston along F2. When the mixture 6+12 has reached the
baffles 19, a back-and-forth movement along F3 and F4 is generated
by a movement of the pistons along F1 and F2, to allow a more or
less rapid passage, and on many occasions, of the said mixture
within the mixing means 15. Typically, the number of mixture return
trips in the card is between one and ten return trips, typically
five return trips to guarantee a satisfactory homogenisation of the
compounds of the reaction. The changes in direction due to the
presence of the baffles thereby allow a good mixing of the dilute
constituents, called thermostable constituents 12, within the
liquid sample of interest 6. Thermostable constituents 12 means in
particular the amplification primers, the detection probe or
probes, the nucleotides and all other thermostable ingredients
required to elongate the primers during the amplification.
[0147] FIG. 9 shows that a first zone exists intended for heating
11 shown by a dotted line. This zone symbolically represents the
place where the manipulator or the robot applies a heat source in
order to treat the liquid sample 6+12, that is to say, the sample 6
in the presence of the thermostable constituents 12, in order to
begin an amplification technique such as NASBA.
[0148] When this is done, FIG. 10 shows that the drawing up along
F2 continues, so that the liquid columns rise to the second
compartment 9 or the non-thermostable constituents 13 are diluted
in turn. This movement along F4 is always due to the rise of the
pistons along F2. Non-thermostable constituents essentially means
the enzymes required for amplification. In the context of a
post-transcriptional amplification, in particular, this means
AMV-RT (Avian Myeloblastosis Virus Reverse Transcriptase), RNase H
and polymerase T7 (DNA dependent RNA polymerase).
[0149] For all the liquid movement steps, the flow rate is
typically 1 .mu.L per second. Advantageously, an optical sensor of
the instrument, not shown in the figures, positioned about two
millimetres before each reagent, loaded in the card, serves to
detect the presence of the liquid segments and thereby to guarantee
satisfactory operating robustness while compensating for the
effects associated with the difference of each sample.
[0150] Alternatively, the use of optical sensors also makes it
possible to measure the length of the liquid segment and therefore
to check the accuracy of the liquid division made during the step
of drawing up and loading the sample in the card.
[0151] In FIG. 11, each liquid column is then relowered along F1 to
the level of the mixing means 15. At this level, the entire mixture
6+12+13 is also moved along F3 and F4, so that the baffles 19 allow
a suitable mixing of the thermostable constituents 12 and
non-thermostable constituents 13 within the sample 6. The arrival
in the chambers can advantageously be detected by the fluorescence
read head built into the instrument to make the real-time
measurement of the amplification reaction.
[0152] FIG. 11, like FIG. 9, shows a second zone for heating 22
which also serves to carry out the NASBA amplification.
[0153] In FIG. 12, the drawing up along F2 enables the liquid
columns 6+12+13 to rise to the third compartment 10, which
accordingly acts as a read zone. It should be noted that this read
zone may be the first compartment 8 or the second compartment 9 or
the third compartment 10. In the present case, the third
compartment 10 acts as a buffer zone to prevent any undesired rise
of liquid towards the top of the device, and this action is
reinforced by the closure of the valve 27.
[0154] If, however, the third compartment 10 acts as a read zone,
the drawing up along F2 is greater, enabling the liquid columns
6+12+13 to rise to the said third compartment 10, where the reading
can take place.
[0155] According to another embodiment, it is possible to provide
for the thermostable constituents 12 to be split into two spheres,
called pellets. A first sphere, present in the first compartment 8,
contains the amplification primers, the nucleotides and any other
thermostable ingredient required for the elongation of the primers
during the amplification. A second sphere, present in the third
compartment 10, contains the detection probe or probes required for
detecting the amplicons, after the amplification step. In this
case, the mixture must return to the level of the mixing means 15,
within which the entire mixture is still moved along F3 and F4 so
that the baffles 19 allow a suitable mixing of the thermostable
constituents 12 and non-thermostable constituents 13 within the
sample 6. The reading can take place in any one of the compartments
8, 9 or 10.
[0156] A final step exists, not shown in the figures, which
consists, immediately after the end of the positioning in the third
compartment, in closing the valve 27 by means of an actuator built
into the instrument. Alternatively, the said valve can be deleted
and replaced by a straw, as mentioned above, which can be sealed,
for example by heat, and which then has two functions, that of
pipetting the sample into the container, and then that of closure
by the use of a heating wire within the associated instrument.
[0157] According to a particular embodiment, a small carousel can
be associated with the device of the present invention. This
carousel carries the various tubes required for carrying out an
extraction step prior to conducting the method according to the
said invention: [0158] the first tube contains the biological
sample to be treated, [0159] at least one second tube contains a
washing buffer, [0160] a third tube for the elution buffer, and
[0161] a fourth tube to recover the eluate. The latter tube is
optional, but useful for taking an aliquot, for example, to perform
a sequencing before a new drawing up into the device for carrying
out the amplification.
[0162] According to this novel embodiment, a silica filter is added
either at the cone 16 or at the pipette cone 28. This silica filter
is available from Akonni (Ref.: 300-10606, Frederick, Md.,
USA).
[0163] According to the method of use, the inventive device
descends to draw up all or part of the biological sample to be
tested (blood, urine, etc.) for about 5 to 100 .mu.L in the first
tube. These values are approximate because they are limited by the
stroke and the volume of the piston (FIG. 1) or pistons (FIG. 3 for
two pistons and FIG. 5 for eight pistons).
[0164] In case of a plurality of pistons, they move simultaneously
to maximise the volume drawn up. Alternatively, the size (stroke
and diameter) of the pistons built into the card can be increased
in order to achieve the best compromise between drawing up a large
volume of sample, on the one hand, and accuracy of movement of the
eluate in the said device, on the other hand, during the
amplification steps.
[0165] The sample is first lysed, optionally in the carousel in the
presence of GuSCn or by ultrasound, in which case the tube is
coupled with a sonotrode placed under the carousel. This sample is
drawn up into the silica filter with, if necessary, return trips to
increase its residence time in the filter and improve the nucleic
acid (RNA/DNA) capture efficiency.
[0166] The remaining sample is then discarded in the first tube or
in another receptacle or tube containing the waste.
[0167] The carousel then rotates to bring the second washing tube
under the pipetting cone. The washing buffer is drawn up by the
pistons, with mixing in the filter if necessary, and then discarded
in the first tube or into another receptacle or tube containing the
waste.
[0168] Optionally, the washing can be carried out at least once
more. In this case, either the device draws up the same washing
buffer as previously, if the latter has not already been used, or
the carousel rotates to bring another second washing tube under the
pipetting cone. The washing procedure is thus repeated with the
washing buffer that is drawn up by the pistons, with mixing in the
filter if necessary, and then discarded in the second tube or in
the waste tube.
[0169] The carousel then rotates to bring the third tube containing
the elution buffer under the pipetting cone. The tube plate may
optionally be provided with a heating block in order to maintain
the temperature of the elution buffer between the ambient
temperature and 75.degree. C., so as to improve the salting out of
the nucleic acids from the filter if necessary.
[0170] A buffer volume of 10 to 160 .mu.l (depending on the
reaction volume per fluid circuit 3 and the number of circuits 3
per device) is drawn up by the pistons, with mixing in the filter
if necessary, and then discarded either in the empty tube after
rotation of the carousel (to recover the eluate) or directly
transferred by drawing up into the card to start the amplification
process.
EXAMPLE
[0171] This example shows the quantification performance obtained
with a disposable device according to the second embodiment of the
invention, that is to say, the card with two fluid channels in
parallel (see FIGS. 3 and 4). The tests were performed using Human
Immunodeficiency Virus (HIV) as target.
Our invention was compared with a product already marketed, called
Nuclisens EasyQ analyzer (Ref. 285060, bioMerieux S.A., Marcy
l'Etoile, France), using the same biological samples, containing
synthetic HIV targets.
1--PREPARATION OF TARGETS
[0172] The transcripts were introduced into the two apparatus:
Nuclisens EasyQ and according to the invention. There was no sample
preparation step, like extraction, for example.
2--MATERIALS AND METHODS
[0173] The experiments on EasyQ were performed using the bioMerieux
HIV2.0 kit (hereinafter called PVB1) (Ref.285033, bioMerieux B.V.,
Boxtel, Netherlands), following the instructions for use. The kit
contained: [0174] a mixture of enzymes: a sphere of enzymes,
hereinafter called ENZ (batch No.: 83281SXX)+45 .mu.L enzyme
diluent (83301AXX), and [0175] a mixture of primers and probes
hereinafter called P/B: there are in fact two P/B spheres (batch
No: 83283SXX), to which 180 .mu.L of diluent were added for P/B 2X
(83272AXX).
[0176] This gave a NASBA mixture with 5 .mu.l of mixture ENZ and 20
.mu.l of mixture P/B+15 .mu.L of targets.
[0177] The inventive disposable device is completely automated for
the amplification and detection. It makes it possible to take the
biological sample to be tested, containing the targets.
[0178] The experimental protocol of the inventive device is defined
for the reagents (primers, probes and enzymes in particular) to
have the same concentration as in the EasyQ protocol. However, the
volume per test used in our invention is 5 .mu.l instead of 40
.mu.l as with EasyQ. The quantity of reagents is therefore divided
by eight. The reagents from the PVB1 kit were freeze-dried and
placed in the inventive device. The freeze-drying bench is
associated with a Hamilton pipettor robot (Ref. 202997, Bonaduz,
Switzerland) which allows the reproducible deposition of droplets
of 1 .mu.L for P/B and 1.25 .mu.L for ENZ in the dedicated
compartments of the inventive device.
[0179] The amplification and detection instrument used with the
invention performs all the functions required to obtain an
amplification curve, that is to say, the drawing up of the sample,
mixing the reagents, heating and fluorescence reading. This concept
eliminates most of the mandatory manual steps with EasyQ.
[0180] Table 1 below shows the main differences between EasyQ and
the invention.
TABLE-US-00001 TABLE 1 Comparison of technical data and of the
method used by the prior art device (EasyQ) and by the invention
EasyQ Invention Volume 40 .mu.L 5 .mu.L P/B 20 .mu.L 1 .mu.L
(incorporated) ENZ 5 .mu.L .sup. 1.25 .mu.L (incorporated) Volume
of 15 .mu.L 5 .mu.L sample used Manual dilute the Load the tube in
the Steps accuspheres, analyzer and start Add primers and probes to
the eluate transfer to the incubator, add the enzyme solution to
the plug, close the tube with the plug, centrifuge, run on the
vortex, centrifuge again, load the tube in the analyzer and start.
Number of Up to 48 tests 2 with the device tests/series
proposed
[0181] As stated above, the biological sample used for this study
contained HIV targets. This sample was then diluted for the
experiments on EasyQ and the invention, but the same series of
dilutions were used. Table 2 below shows the number of HIV targets
per test and the number of experiments performed with each of the
two devices (EasyQ and invention). The number of pre-extraction
"equivalent" copies corresponds to the number of copies needed
upstream of an extraction step to obtain the number of copies per
test (i.e. pre-extraction "equivalent" is equal to the number of
copies per test divided by the extraction yield).
TABLE-US-00002 TABLE 2 Number of HIV targets per test and number of
experiments performed with each of the two devices (EasyQ and
invention) Number of copies of target per test (EasyQ and
invention) 0 3.75 7.5 15 22.5 30 300 3000 Number of 0 12.5 25 50 75
100 1000 10 000 copies of pre- extraction "equivalent" per test
Number of 15 12 12 15 12 15 15 15 replicates with EasyQ Number of 8
8 8 4 4 4 4 4 replicates with the invention
[0182] "Replicate" means the number of times that the test was
performed in parallel using the same initial sample. The number of
copies for internal inspection was 290 per test, both for EasyQ and
for the invention. Note that in the present case, the invention
does not allow two tests in parallel, so that the number of
replicates is considerably lower with our invention than with
EasyQ.
[0183] The detection limit claimed for EasyQ is 25 copies
(pre-extraction equivalent), corresponding to 7.5 copies per
test.
[0184] The data acquired with EasyQ were processed with the EasyQ
Director software (BioMerieux S.A., La Balme, France), using the
HIV-1DB 2.0 test protocol (Ref. 285033, bioMerieux B.V., Boxtel,
Netherlands). Each amplification curve, measured with the
instrument using the invention, was processed by using an internal
tool like EDrecalc recalculation concerning the algorithm for
computation and interpretation of the amplification curves, which
is included in the EasyQ Director commercial software mentioned
above, with the same algorithm as the one used by the EasyQ
Director software and the HIV-1DB 2.0 test protocol.
3--RESULTS
[0185] The raw fluorescence curves are shown in FIG. 13 for the
prior art device (EasyQ) and in FIG. 14 for the invention. The
amplification curves obtained with the invention are very similar
to those obtained with EasyQ. A broad distribution of fluorescence
values can be observed for the fluorescence plateau of the signal
from the calibrator. Since this distribution exists with each
instrument, this propagation is not associated with the
instrumentation.
[0186] 3.1--Detection Limit:
[0187] Owing to the small number of replicates, it is not possible
to determine the detection limit with a narrow confidence interval.
Thus, in Table 3 below, we have only compared the number of
positive results obtained with the two instruments for a few values
of the number of copies extracted from Table 2:
TABLE-US-00003 TABLE 3 Detection limit with each of the two devices
(EasyQ and invention) Input values (pre-extraction "equivalent"
copies per test) 0 12.5 25 50 75 Easy Q 0/15 6/12 12/12 14/15 12/12
Number of positives/number of experiments Invention 0/8 5/8 8/8 4/4
4/4 Number of positives/number of experiments
[0188] The detection limit claimed for the NucliSENS HIV 2.0 test
is 25 copies (detection limit at 95% positives). With this input
value, all the tests performed with the inventive device were
positive. With 12.5 copies, about 50% of the tests were positive
with EasyQ and slightly more with the invention. It can therefore
legitimately be considered that our invention has results at least
similar to the detection limit of the prior art, EasyQ.
[0189] 3.2--Quantification Performance:
[0190] FIG. 15 shows the Qratio (quantification variable) as a
function of the number of input copies. The distribution of the
points corresponding to the data is similar for both
instruments.
[0191] Using the parameters associated with the reagent batch, a
person skilled in the art can obtain the number of copies by
calculation, and the mean thereof is plotted on a logarithmic scale
in FIG. 16.
[0192] Tables 4 and 5 below show the qualification performance
associated with these two instruments:
TABLE-US-00004 TABLE 4 Qualification Performance for EasyQ Input
Log Degree of Data (Input) Mean Accuracy Accuracy 25 1.40 1.31 0.19
0.09 50 1.70 1.40 0.20 0.30 75 1.88 1.64 0.19 0.24 100 2.00 1.70
0.23 0.30 1000 3.00 2.75 0.14 0.25 10 000 4.00 3.83 0.07 0.17
TABLE-US-00005 TABLE 5 Qualification Performance for the Invention
Input Log Degree of Data (Input) Mean Accuracy Accuracy 25 1.40
1.47 0.18 0.07 50 1.70 1.34 0.33 0.36 75 1.88 1.77 0.05 0.11 100
2.00 1.82 0.34 0.18 1000 3.00 2.89 0.21 0.11 10 000 4.00 3.84 0.13
0.16
[0193] According to the high level specifications of the HIV2.0
test, the accuracy, that is to say, the standard deviation of the
results of the various replicates, must be lower than 0.3 log. Some
accuracy values for the data of the instrument prototype associated
with the device are above this specification. This may be due to
the small number of replicates, which preclude a correct estimation
of the accuracy.
[0194] The prototype clearly meets the specification for the degree
of accuracy (that is to say, the difference between the result and
the number of test input copies) is 0.25.
[0195] The linearity for the invention is the same as for EasyQ,
but needs to be measured above the 10.sup.4 copies of this
study.
4--CONCLUSION
[0196] The present invention, in its configuration with two
parallel fluid channels, was used to detect HIV targets by means of
the HIV v.2.0 kit. The results were compared with the EasyQ
analyser, which served as a reference.
[0197] The performance of the invention was in line with the most
severe constraints required for performing an HIV test (HIV v.2.0)
and are at least comparable to those recorded with the EasyQ
analyser.
[0198] Since the invention used a reaction volume of 5 .mu.L
(instead of 40 .mu.L for EasyQ), the quantity of the reagents per
test was divided by eight. This gives rise to a much lower
production cost per test, because this cost is mainly due to the
enzymes, accounting for about 80% of all the ingredients in the
kit. The inventive device also significantly reduces the number of
manual steps, because only three basic actions are required to
launch a test: [0199] Loading of the inventive device; [0200]
Loading the tube containing the extracted targets (by the EasyMAG
extraction apparatus (Ref.200111, bioMerieux SA, Marcy l'Etoile,
France)), and [0201] Starting the test.
REFERENCE NUMERALS
[0201] [0202] 1--Disposable device [0203] 2--Solid body of the
device 1 [0204] 3--Fluid circuit or channel in the body 2 [0205]
4--Through hole of channel called inlet [0206] 5--Through hole of
channel called outlet [0207] 6--Liquid and biological sample of
interest [0208] 7--Drawing up/discharging means [0209] 8--First
compartment along channel 3 [0210] 9--Second compartment along
channel 3 [0211] 10--Third compartment along channel 3 [0212]
11--First zone intended for heating [0213] 12--Thermostable
constituents present in compartment 8 [0214] 13--Non-thermostable
constituents present in compartment 9 [0215] 14--Boundary film
[0216] 15--Mixing means [0217] 16--Built-in pipette cone or spindle
accommodating a cone 28 [0218] 17--Cylinder of the drawing
up/discharging means 7 [0219] 18--Piston of the drawing
up/discharging means 7 [0220] 19--Baffle of the mixing means 15
[0221] 20--Container in which the sample 6 is initially present
[0222] 21--Means of linkage with an actuator [0223] 22--Second zone
intended for heating [0224] 23--Guide of piston 18 [0225]
25--Through hole [0226] 26--Piston head [0227] 27--Final closure or
sealing valve [0228] 28--Conventional pipette cone [0229]
100--Disposable device with two parallel channels 3 [0230]
200--Disposable device with eight parallel channels 3 [0231]
F1--Movement of piston 18 in cylinder 19 which decreases the volume
of fluid in fluid circuit 3 [0232] F2--Movement of the piston 18 in
the cylinder 19 which increases the volume of fluid in fluid
circuit 3 [0233] F3--Movement of the sample 6 under the action of
movement F1 [0234] F4--Movement of the sample 6 under the action of
movement F2 [0235] F5--Socketing of cone 28 on sleeve 16
* * * * *