巴西专利BR112014007440B1 unified reagent slide and methods for detecting the presence or absence of a pipette tip within a pi

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专利摘要:
Unified Reagent Slide and Methods for Detecting the Presence or Absence of Pipette Tip Within Pipette Sheath and For Determining Pipette Tip Length Within Pipette Sheath. The embodiments disclosed in this document refer to unified reagent slides to contain and transport reagents and materials used in the automatic preparation and / or processing of samples for biological and / or chemical tests.
公开号:BR112014007440B1
申请号:R112014007440-2
申请日:2012-09-28
公开日:2020-11-03
发明作者:Ammon David Lentz；Dwight Livingston；Adam Bruce Steel；Richard St. Pierre
申请人:Becton, Dickinson And Company；
IPC主号:

专利说明:

DESCRIPTIVE REPORT REMISSIVE REFERENCE TO RELATED ORDERS
[001] This Order claims priority to Serial US Provisional Order No. 61 / 541,991, entitled “UNITIZED REAGENT STRIP'e filed on September 30, 2011, the disclosure of which is incorporated herein in full by reference . BACKGROUND Technical Field
[002] In general terms, the technology described in this document refers to supports for reagents and disposable products that can be used to transport reagents and perform processing operations with the reagents, for example, in automated sample processing / preparation devices Background
[003] The automation of diagnostic tests and high-throughput screening has become more prevalent, and various devices have been developed to meet the growing need for fast, delicate and consistent analyzes of various samples. For example, in recent years, integrated devices have been developed for sample preparation and processing, for example, for nucleic acid assays.
[004] Many important assays require the isolation of various components, such as nucleic acids, proteins and the like, from clinical and / or environmental samples. The isolation of nucleic acids, proteins and other analytes of interest from clinical or environmental samples can be time-consuming and laborious. Manual sample preparation is also subject to greater variation due to inaccuracies and human errors. Several variables influence the consistency and precision during sample preparation, which usually involves several agents and the need for several transfer operations (for example, pipetting). Often, the desired agents are of sufficient variety that they typically require different handling in relation to each other and are available from different suppliers. Therefore, the variation between different suppliers and lots of a specific reagent, and the different handling of several reagents by one or more individuals, can lead to variability in the assay. In addition, several pipetting operations introduce the possibility of cross-contamination, for example, between samples or within samples (for example, the reagents used during different stages of preparation and / or processing of the same sample).
[005] There is a need for methods and devices to prepare and process large numbers of samples in parallel and which minimize the variability between assays. Desirably, the methods and devices would minimize the user's manipulation of disposable agents and / or products used in the preparation and processing procedures to allow efficient processing of a sample, minimize contamination and imprecision and maintain flexibility .
[006] The discussion of the background of the invention in this document is included to explain the context of the inventions described in this document. It should not be construed as acknowledging that any material referenced has been published, known or is part of common knowledge by the priority date of any of the Claims. BACKGROUND
[007] Unified reagent slides and methods for using them are revealed in this document. In one aspect, a unified reagent slide is revealed, which comprises: a slide with an upper face and a lower face, which comprises: a first and a second pipette sheath with opposite sides, said first pipette sheaths and second comprising a first and a second pipette tip openings, respectively, each of which consists of a distinct opening in the upper face of the blade, wherein said first and second pipette tip openings are configured for the introduction of tips of first and second pipette in said first and second pipette sheaths, respectively, and wherein each of said first and second pipette sheaths is configured to substantially encircle the length of the first and second pipette tips, respectively; a process tube; and a receptacle, which comprises an opening through the reagent slide, wherein said receptacle is configured to receive a reagent tube.
[008] In another aspect, this document reveals a method for detecting the presence or absence of a pipette tip inside a pipette sheath in a unified reagent slide, which comprises: a slide with a top face and a bottom face, which comprises: a first and a second pipette sheaths with opposite sides, said first and second pipette sheaths comprising a first and a second pipette tip openings, respectively, each of which consists of an opening distinct on the upper face of the slide, in which said first and second pipette tip openings are configured to insert first and second pipette tips into said first and second pipette sheaths, respectively, and in which each of said sheaths pipette first and second is configured to substantially encircle the length of the first and second pipette tips, respectively; a process tube; and a receptacle, which comprises an opening through the reagent slide, wherein said receptacle is configured to receive a reagent tube, wherein said first pipette sheath comprises a pair of openings, said pair of openings comprising a first orifice with core and a second orifice with core, which cross the side wall of the first pipette sheath, where the holes with first and second core are formed on opposite sides of the side wall of the first pipette sheath and are located at the same distance along the length of the first pipette sheath from the first pipette tip opening; emitting an optical beam through the first core hole of said pair of openings in the pipette sheath; and detecting whether said optical beam leaves unobstructed through the second hole with the core of said pair of openings of the first pipette sheath, wherein the unobstructed exit of said optical beam through said second hole with the core of said first pipette sheath indicates the absence of a pipette tip within the pipette sheath and in which the obstructed outlet of said optical beam through the second core hole indicates the presence of a pipette tip within said first pipette sheath.
[009] In another aspect, this document discloses a method for determining the length of a pipette tip within a pipette sheath on a unified reagent slide, the method comprising: providing a unified reagent slide, which comprises : a blade with an upper face and a lower face, said blade comprising: a process tube; a receptacle, which comprises an opening through the reagent slide, wherein said receptacle is configured to receive a reagent tube; a first and a second pipette sheath, each of said pipette sheaths comprising: a first and second pipette tip openings, respectively, each of which consists of a distinct opening in the upper face of the slide, wherein said first and second pipette tip openings are configured to insert first and second pipette tips into said first and second pipette sheaths, respectively, and wherein each of said first and second pipette sheaths are configured to substantially surround the length of the first and second pipette tips, respectively; a pair of upper openings of the pipette sheath and a pair of lower openings of the pipette sheath in said first pipette sheath, each of said pairs of upper and lower openings comprising a first and a second core holes, which pass through the side wall of the first pipette sheath, wherein the first and second core holes of said pairs of upper and lower pipette sheaths are formed on opposite sides of the first pipette sheath and are located at the same distance along the length of the first pipette sheath in relation to the first pipette tip opening, and wherein said pair of upper pipette sheath openings is closer to the first pipette tip opening than said pair of lower pipette sheath openings ; emitting an optical beam through said first orifice with core of said pair of upper openings of the pipette sheath; emitting an optical beam through said first orifice with core of said pair of lower openings of the pipette sheath; detecting whether said optical beam has been prevented from passing through the second core hole of said pair of upper openings in the pipette sheath; and detecting whether said optical beam has been prevented from passing through said hole with the lower core of said pair of openings of the first pipette sheath, wherein the obstruction of said optical beam through the second hole with the core of the pair of upper openings and the passage of said optical beam through said second orifice with core of said pair of lower openings in the pipette sheath indicate that the pipette tip within said first pipette sheath has a length that does not extend to the pair of lower openings in the sheath pipette when inserted into the first pipette sheath.
[0010] In yet another aspect, a method is revealed for determining the length of a pipette tip within a pipette sheath on a unified reagent slide, the method comprising: providing a unified reagent slide, which comprises: a blade with an upper face and a lower face, said blade comprising: a process tube; a receptacle, which comprises an opening through the reagent slide, wherein said receptacle is configured to receive a reagent tube; a first and a second pipette sheath, each pipette sheath comprising: a first and a second pipette tip openings, respectively, each of which consists of a distinct opening in the upper face of the slide, wherein said tip openings first and second pipette tips are configured to insert first and second pipette tips into said first and second pipette sheaths, respectively, and wherein each of said first and second pipette sheaths is configured to substantially encircle the length of the tips pipette first and second, respectively; a pair of upper openings of the pipette sheath and a pair of lower openings of the pipette sheath in said first pipette sheath, each of said pairs of upper and lower openings comprising a first and a second core holes, which pass through the side wall of the first pipette sheath, wherein the first and second core holes of said pairs of upper and lower pipette sheaths are formed on opposite sides of the first pipette sheath and are located at the same distance along the length the pipette sheath in relation to the pipette tip opening, and wherein said pair of upper pipette sheath openings is closer to the first pipette tip opening than said pair of lower pipette sheath openings; emitting an optical beam through said first orifice with core of said pair of upper openings of the pipette sheath; emitting an optical beam through said first orifice with core of said pair of lower openings of the pipette sheath; detecting whether said optical beam has been prevented from passing through the second core hole of said pair of upper openings in the pipette sheath; and detecting whether said optical beam has been prevented from passing through said hole with the lower core of said pair of openings of the first pipette sheath, wherein the obstruction of said optical beam through the second hole with the core of the pair of upper openings and the passage of said optical beam through said second orifice with core of said pair of lower openings in the pipette sheath indicate that the pipette tip within said pipette sheath has a length that does not extend to the pair of lower openings in the pipette sheath when inserted into the pipette sheath.
[0011] In yet another aspect, a method is disclosed in this document to determine the length of a pipette tip within a pipette sheath on a unified reagent slide, the method comprising: providing a unified reagent slide, which comprises : a blade with an upper face and a lower face, said blade comprising: a process tube; a receptacle, which comprises an opening through the reagent slide, wherein said receptacle is configured to receive a reagent tube; a first and second pipette sheaths, each comprising: a first and a second pipette tip openings, respectively, each of which consists of a distinct opening in the upper face of the slide, wherein said pipette tip openings first and second are configured to insert first and second pipette tips into said first and second pipette sheaths, respectively, and wherein each of said first and second pipette sheaths are configured to substantially encircle the length of the pipette tips first and second, respectively; a hole with an upper core and a hole with a lower core in said first pipette sheath, each of said holes with an upper and lower core crossing the side wall of the pipette sheath, wherein said hole with an upper core is closer to the first pipette tip opening than said orifice with lower core; determining whether a pipette tip extends within said first pipette sheath from the first pipette tip opening to the distance from the first core hole; and determining whether a pipette tip extends into said first pipette sheath from the first pipette tip opening to the distance from the second core hole. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The one or more several modalities presented in this document are described in detail with reference to the following Figures. The drawings are provided for purposes of illustration only and merely represent typical or exemplary embodiments of the invention. These drawings are provided to facilitate the understanding of the invention by the reader and should not be interpreted as restricting the breadth, scope or applicability of the invention. It is worth noting that, for the sake of clarity and ease of illustration, these drawings are not necessarily proportional to the actual dimensions.
[0013] Some of the Figures included in this document illustrate various embodiments of the invention viewed from different angles. Although the accompanying descriptive text refers to these views as “top”, “bottom” or “side” views, these references are purely descriptive and do not necessarily imply or require that the invention be implemented or used in any specific spatial orientation, unless explicitly stated otherwise.
[0014] These and other characteristics and advantages of the various modalities revealed in this document will be better understood with reference to the following description and drawings, in which equal reference numbers refer to equal parts throughout them and among which:
[0015] FIG. IA is a perspective view of a reagent slide as described in this document.
[0016] FIG. 1B is a perspective view of the reagent slide as described in this document, with a separate reagent tube (160) illustrated as inserted into the slide.
[0017] FIG. 1C is a sectional view of the process tube along the cut line A-A in FIG. IA.
[0018] FIG. 1D is a sectional view of the reagent tube 140 along the cut line B-B in FIG. IA.
[0019] FIG. 1E is a cross-sectional view of the pipette sheath along cut line C-C in FIG. IA.
[0020] FIG. 1F is a top view of the reagent slide of FIG. IA.
[0021] FIG. 1G is a bottom view of the reagent slide of FIG. IA.
[0022] FIG. 1H is a cross-sectional view of one embodiment of the reagent slide of FIG. IA.
[0023] FIG. 2A is a perspective view of a reagent slide as described in this document.
[0024] FIG. 2B is a top view of the reagent slide of FIG. 2A.
[0025] FIG. 2C is a bottom view of the reagent slide of FIG. 2A.
[0026] FIG. 3 is a plan view of a reagent slide as described in this document.
[0027] FIGS. 4A to 4E illustrates a sequence of pipetting operations when using a laminated layer.
[0028] FIGS. 5A and 5B illustrate modalities of a laminated layer. DETAILED DESCRIPTION OF THE PREFERRED MODE
[0029] The modalities described in this document propose reagent holders configured to contain, transport and store various reagents and materials used in the preparation and processing of samples, for example, clinical and / or environmental samples. The reagent holders proposed in this document have several advantages for sample preparation and processing, such as clinical and / or environmental samples, and are suitable for use with automated sample processing devices. As an example, some of the advantages provided by the reagent holders disclosed in this document include, among others, a model that (1) minimizes the cross contamination of reagents and samples; (2) facilitates the quality control of blades / disposable products; (3) simplifies manufacturing; and (4) offers useful versatility for different molecular platforms and automated devices.
[0030] The supports disclosed in this document are also configured for use by devices that carry out automated sample preparation, for example, of several samples at the same time. An exemplary form of such an apparatus is described, for example, in the International Patent Application Publication in WO 09/054870, which is incorporated by reference in its entirety into this document.
[0031] The preparation of a sample for use in assays, such as nucleic acid testing ("NAT"), for example, by polymerase chain reaction ("PCR") or the like, may include one or more of the following steps: placing a sample of polynucleotides in contact with a mixture of reagents in order to perform the nucleic acid test (“NAT”), for example, in the case of a polymerase chain reaction (“PCR”) or of another amplification comprising a polymerase enzyme and several nucleotides. In some embodiments, the reagent mixtures further comprise hybridization probes with detectable clusters, where the probes hybridize specifically to target nucleic acids (and / or target nucleic acid sequences by positive control).
[0032] In some embodiments, the reagent mixture may be in the form of one or more lyophilized granules, as stored in a reagent tube in the holder, and the method may also include reconstituting the one or more reagent granules with liquid to create a solution mixing reagents for PCR. The support disclosed in this document provides, in a self-contained manner, all the reagents necessary to prepare a sample of nucleic acid ready for testing, or, when supplied to the user in the form of a kit, contains, together with other packages, all reagents needed. Suitable reagents and protocols for using them in DNA and RNA extractions can be found, respectively, in US Patent Application Publications No. US 2010-0009351 and US 2009-0131650, each of which is incorporated into this document by reference.
[0033] Various characteristics of the reagent holders described in this document are described with reference to the accompanying drawings. Each of the exemplary supports illustrated in FIGs. from AI to 2H, from 2A to 2C and 3 can be called a "unified disposable blade" or "unified blade" because they are intended for use in the form of a single unit configured to contain all reagents and receptacles necessary to prepare a sample and because they are designed in the shape of a blade. It is consistent with the description in this document, however, that other geometric structures for the various receptacles are used, in such a way that the description is not limited to a linear or blade structure, but may include a circular or network structure.
[0034] Referring to Figures 1 to 3, exemplary reagent slides 100 are illustrated. A reagent slide 100 comprises a slide 110, which has an upper face 310 and a lower face 320, which houses various components used in the preparation and / or sample processing, including one or more pipette sheaths 120 and one or more process tubes 130, and which also houses one or more integrated reagent tubes 140 with reagent tube openings 330. In some embodiments, reagent tubes 140 they are integrated / integral with blade 110. In some embodiments, process tubes 130 are integrated with blade 110. In some embodiments, process tubes 130 are separate parts from the unified blade. In some embodiments, the reagent slide comprises one or more tube receptacles 150. Tube receptacles 150 can be integrated / integral with slide 110 and are configured to receive one or more reagent tubes 160, which are not integrated / integral with the slide 110. In some embodiments, reagent tubes 160 can be integrated into the slide, as shown in FIG. 2A.
[0035] As an example, the unified reagent slides, as described in this document, may include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pipette sheaths, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more process tubes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more receptacles, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more integrated reagent tubes, 1, 2, 3, 4, 5 or more waste containers, or the like, arranged in any configuration on the slide.
[0036] In preferred embodiments, the reagent slide comprises one or more pipette sheaths 120 substantially separate from adjacent pipette sheaths and / or from reagent tubes 140, process tubes 130 or adjacent tube receptacles 150. Preferably, pipette sheaths 120 are integrated with slide 110 and therefore do not require manual assembly on slide 110. Individual pipette tips can be inserted into each individual pipette sheath 120, thanks to individual pipette tip openings 170 present in slide 110. The pipette sheaths 120 substantially surround the sides and the base of the individual pipette tips. The term "substantially encircle", when used with reference to pipette sheaths, means that a sheath surrounds at least the main body of a pipette tip. That is, the top of the pipette tip may comprise a nozzle, or something like that, (through which the pipette is inserted), which extends beyond the top of the slide 110 (and possibly rests on top of it). In some embodiments, the pipette sheath surrounds, for example, 70%, 80%, 85%, 80%, 90%, 95% or more the length of a pipette tip. When substantially surrounding individual pipette tips, pipette sheaths prevent contact between each pipette tip and other pipette tips, reagent tubes, process tubes, waste containers or the like present on the slide. More specifically, each pipette sheath is configured to contain material that surrounds or forms a barrier or wall 290 that insulates the body from a pipette tip inserted in it from other reagents / supports or disposable products (for example, from other pipette) on the unified slide. Therefore, individual pipette sheaths prevent cross-contamination between reagents and / or samples handled during pipetting and / or preparation. For example, pipette sheaths 120 prevent contamination between adjacent pipette tips on the same slide, as well as between pipette tips housed in adjacent reagent tips, for example, within an automated sample preparation / processing device.
[0037] In some embodiments, the pipette sheaths contain one or more sheath openings or 180-core holes. In some embodiments, the 180-core holes are present in the form of pairs of sheath openings, whereas in in other embodiments, the core holes are not part of a pair of openings. In some embodiments, the pipette sheaths comprise one, two, three, four, five, six, seven, eight, nine, ten or more orifices with a peerless core 180. In some embodiments, the pipette sheaths comprise several pairs of openings , each of which comprises two 180-core holes. For example, a pipette sheath may include one pair, two pairs, three pairs, four pairs, five pairs, six pairs, seven pairs, eight pairs, nine pairs, ten pairs or more pairs of openings in it. A pair of openings in a pipette sheath comprise a first orifice with core 180a and a second orifice with core 180b, which are formed from opposite sides and at the same distance from the top of the pipette sheath 120, as illustrated, for example , FIG. 1 AND. In some embodiments, holes with an unpaired core 180 are formed on opposite sides, but at varying distances from the top of the pipette sheath 120.
[0038] Orifices with core 180, whether unique or arranged in the form of one or more pairs of openings, can, to the greatest advantage, be used to determine the presence or absence of a pipette tip within a pipette sheath 120 , either manually (by visual inspection) or automatically (for example, by an optical sensor). In this way, the 180-core holes provide an additional quality control checkpoint before using the unified reagent slide. For example, in the context of automated detection of pipette tips, when two orifices with core 180 take the form of a pair of openings in the pipette sheath, light is emitted through the first orifice with core of that pair. When the pipette sheath 120 does not contain a pipette tip, the light passes through the first and second core holes of the pair of aligned openings formed on opposite sides of the sheath. When a pipette tip is present inside the sheath, the pipette tip blocks or obstructs the visible path between the first and second core holes of each pair of openings. Thus, pairs of sheath openings 180 can be readily used to determine whether or not a pipette tip is present in each sheath 120. When holes with core 180 are present, but not as part of a pair of sheath openings of the pipette, it is possible to determine the presence or absence of a pipette tip inside the sheath by calculating, for example, the reflection or obstruction of the light emitted through the hole with an unmatched core, since the reflection or obstruction will be different if a tip pipette is present or absent within the pipette sheath. For example, in some embodiments, the detection of the reflection of light can be determined using means and devices recognized in the art as retroreflective detectors. In some embodiments, the presence or absence of a pipette tip in a sheath is determined by measuring light obstruction, for example, using means and devices recognized in the art, such as through-beam sensors.
[0039] As mentioned, in some embodiments, more than one pair of pipette sheath openings 180 is present in the sheath, as illustrated, for example, in FIG. 1 AND. When multiple orifices with core 180 are formed in a pipette sheath (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), each orifice with core is formed in one position or one different distances along the length of the pipette sheath in relation to the upper face of the slide 110, which defines the pipette tip opening. Similarly, several pairs of sheath openings 180 can be formed along the length of the same pipette sheath, each pair of sheath openings 180 arranged in a different position or distance along the length of pipette sheath 120 in relation to the upper face of the slide 110, which defines the pipette tip opening. The distribution of several 180-core holes along the length of a pipette sheath (either without a pair or as part of a pair of pipette sheath openings) makes it possible not only to determine whether or not a pipette tip is present within the sheath, but also determine the length (size) of a pipette tip inserted in the sheath. For example, when a shorter pipette tip is present in sheath 120, it may alter the reflection or obstruction of the light emitted through the orifice 180a but may be too short to alter the reflection or obstruction of the light emitted through the 180d core hole. Likewise, when several pairs of sheath openings are present, a tip present in the pipette sheath can obstruct the passage of light emitted through the orifice 180a as it leaves the orifice 180b but may be too short to obstruct the light that passes through the pairs of sheath openings 180c-180d or 180e-180f. Pairs of pipette sheath openings 180, therefore, have advantages in the quality control of the reagent slides because they allow to quickly determine the presence or absence of tips, which can be done manually (for example, by visible inspection by an individual ) or can be readily automated during the manufacturing process or the reagent slide assembly process. For example, it is possible to use optical sensors to transmit and detect the light that enters or leaves the first or second core holes of the pairs of openings 180 in order to detect the presence or absence (and, for example, the length) of pipette inside each individual sheath 120.
[0040] In addition to bringing advantages to quality control, holes with core 180 can facilitate the manufacture of reagent slides 100. More specifically, the manufacture of long and relatively narrow sheaths, such as pipette sheaths 120, by injection molding brings significant challenges. Pipette sheaths are usually long and narrow in shape with little angled vessels. Long center pins, which are conventionally used in injection molding of structures such as pipette tip sheaths as described in this document, tend to move with high pressure injection, for example, of the thermoplastic or thermoset material from which the blades of reagents are made. The presence of 180-core holes, either without pairs or as part of pairs of sheath openings, allows the use of stabilizing pins, produced by molding, to be used in order to stabilize the long central pins used for molding the sheaths. pipette 120. Therefore, the holes with core 180 enable the manufacture of unified pipette sheaths by injection molding, thus simplifying and reducing manufacturing costs.
[0041] In some embodiments, the reagent slide comprises pipette sheaths of the same length. In some embodiments, the reagent slide may comprise pipette sheaths of different lengths. For example, as shown in FIG. 2, the reagent slide 100 comprises one or more pipette sheaths 140 with a first length 230 and one or more pipette sheaths with a second length 240, as illustrated, for example, in FIG. 2A. Therefore, pipette sheaths with the first length 230 may have longer pipette tips than pipette sheaths with the second length 240, which are shorter. As discussed above, in some embodiments, the pipette sheaths comprise one or more orifices with unpaired core 180 or holes with a core in the form of pairs of pipette sheath openings. In some embodiments, however, the pipette sheaths do not comprise a 180-core hole. In some embodiments, for example, a reagent slide is proposed that comprises one or more pipette sheaths with a first, longer, and longer length. pairs of pipette sheath openings along that longest length; and one or more pipette sheaths with a second, shorter length, and no orifice with core or pair of pipette sheath openings. In some embodiments, however, both the longest and the shortest pipette sheaths comprise at least one unpaired core hole 180 or at least one pair of pipette sheath openings. In some embodiments, the longer pipette sheath comprises more orifices with an unpaired core 180 or pairs of pipette sheath openings than the shorter pipette sheath. As an example, a shorter pipette sheath may contain one or two holes with a 180 core, or one or two pairs of pipette sheath openings, and a longer pipette sheath may contain three or four holes with an unmatched core 180, or three or four pairs of pipette sheath openings.
[0042] As shown in FIG. 3, the pipette sheaths 120 are closed at the base, which provides space to collect any liquids or drips from the pipette tips after use. The individual pipette sheaths are substantially separated, for example, by a wall.
[0043] As discussed above, the reagent slides disclosed in this document preferably comprise one or more receptacles 150. The reagent slide receptacles 150 can be configured to receive reagent tubes that contain, respectively, sufficient amounts of one or more reagents used to prepare and / or process biological and / or environmental samples. In some embodiments, the reagents may be in solid form, such as in lyophilized form, to perform sample preparation and / or processing, for example, isolating nucleic acids from a sample to create a suitable sample for the nucleic acids (“NAT”) associated with the support. In some embodiments, the reagents may be in liquid form.
[0044] The one or more receptacles 150 may be of the same size and shape, or they may be of different sizes and shapes from each other. The receptacles 150 are illustrated with open bases, but are not limited to this topology, and can be closed, except for the entrance 220 in the upper face of the blade 110. Preferably, the receptacles 150 are configured to receive containers, vases or tubes in common use in the field of laboratory analysis, or containers suitably configured for use with the support disclosed in this document. Therefore, the reagent tubes 160, which are not integrated with the slide 110, can be stored apart from the reagent slides and fitted into them immediately before use. This is advantageous because different reagents (for example, reagents for extraction of nucleic acids vs. reagents for PCR) may require different storage conditions. For example, lyophilized reagents may be sensitive to moisture and require different storage conditions, for example, from a lysis buffer. The model with quick fitting of reagent tubes also brings versatility, since tubes containing different reagents can be mounted on the reagent slide 100 depending on the different types of preparation / processing that the user wants to perform with the sample.
[0045] The blades disclosed in this document may include a front edge 190. The front edge 190 can be configured to facilitate handling by the user. The leading edge 190 can also be configured to facilitate the proper introduction and / or positioning of the reagent slide 100, for example, in an automated preparation and processing device. The front edge 190 may contain certain identification features, such as color, barcode, RFID or the like, to facilitate the identification and / or monitoring of individual reagent slides 100.
[0046] In some embodiments, the reagent slide 100 comprises a positioning member, such as a mechanical key 250. Typically, this key is part of the slide 100, for example, part of the leading edge 190 or the like. A mechanical key guarantees the reception of the support in a complementary member, for example, in a support rack or in a receiver compartment of an apparatus that controls the pipetting operations on the reagents in the support. A mechanical key 250 is usually a cutout of a specific shape that matches a corresponding cutout or protuberance on a receiving device. Thus, the reagent slide 100 may comprise a mechanical key 250 that consists of a pair of rectangular cutouts at the end of slide 110. This feature, as illustrated, also provides a tab through which the user can obtain a suitable mechanical advantage for inserting and remove the support from a rack or other device. Those skilled in the art will realize that the location of the mechanical key 250 feature may be different from that illustrated in the attached Figures. For example, the mechanical key 250 can be located on the other end of the blade 110, other than the front edge 190. In some embodiments, the key 250 is an angled cutout that facilitates the introduction of the support in a rack, as well as ensuring placement in the rack when placing a complementary angled cutout in a recessed area configured to receive it. Other variations of a mechanical key are, of course, consistent with the description in this document: for example, curved cutouts or various combinations of notches or protuberances, all would facilitate the safe positioning of the support.
[0047] In some embodiments, the reagent slide comprises an identifier attached to slide 100. The identifier can be a label, such as a writable label, a bar code, a two-dimensional bar code or an RFID tag. The identifier serves, for example, to quickly reveal which combination of reagents is present in the holder and, thus, what type of sample preparation protocol is intended. The identifier can also indicate the batch from which the support was produced, for the purposes of quality control and record keeping. The identifier can also allow the user to correlate specific support to a specific sample.
[0048] As discussed above, reagent tubes 140 and 160, such as containing the lyophilized reagents, can be sealed at the top by a metallic foil, such as an aluminum foil, without any layer of plastic lining, as described in more detail in this document. Reagent tubes 160 containing reagents can be supplied in the form of single tubes or several tubes with completely different vessels, in which the vessels are joined, for example, by means of a connector. For example, in some embodiments, more than one reagent tube 160 (for example, two, three, four, five, six, seven, eight, nine, ten or more) can be supplied together, in which the reagent tubes are fitted together in place in adjacent 150 receptacles. In the example guide, several reagent tubes 160 containing specific reagents for a specific NAT assay (for example, containing lyophilized amplification primers and / or control probes and / or nucleic acids) can be joined together and readily fitted into a slide 110 configured to receive the various different reagent tubes joined together. In other embodiments, the receptacles are configured so that it is possible to insert reagent tubes 160 individually into each receptacle 150.
[0049] Integrated reagent tubes 140 and / or quick-fit reagent tubes 160 containing different reagents can be of different colors or color coded to facilitate identification by the user. For example, integrated colored reagent tubes 140 can be useful for distinguishing different types of unified reagent slides, for example, which can be used in different sample preparations. In the case of quick-fit reagent tubes 160, color coding of the tubes can be used to distinguish different reagents from each other. By way of example, in the case of unified reagent slides used for DNA isolation and the generation of a PCR-ready sample, different reagent tubes color coded 160 can be used to distinguish tubes used with respect to different NATs, for example , with different pairs of oligonucleotide primers, and the like. For example, they can be made of different colored materials, such as dyed plastic, or they can have some kind of identifying mark on them, such as a colored stripe or dot. They may also have a label printed on the side and / or may have an identifier, such as a barcode, on the seal layer at the top. In some embodiments, process tubes 130 and / or reagent tubes 140 and 160 can be translucent.
[0050] The reagent slides 100 are illustrated with a waste chamber 200, which includes an opening for the entry of waste 210 in the upper face of the slide 110. The waste chamber 200 is optional and, in the modalities in which it is made present, it is configured to receive used liquid reagents. In other modalities, where used liquid reagents are not present, they are transferred and disposed of in a place outside the support, such as, for example, a sample tube that contained the original sample whose content was analyzed. The waste chamber 200 is illustrated as part of a set that also comprises two or more reagent tubes 140. It should be borne in mind that this structure is constructed for convenience, for example, manufacturing purposes; other locations of the waste chamber 200 are possible, such as modalities in which the waste chamber 200 is adjacent to a reagent tube 140, but not connected to it except by slide 110.
[0051] The support is typically such that the slide 110, the one or more pipette sheaths 120, the process tube 130, the two or more reagent tubes 140 and the waste chamber (if present) are made of one piece , made of a material such as polypropylene. As discussed above, the model of the modalities disclosed in this document advantageously facilitates the manufacture of a unified reagent slide, for example, by injection molding.
[0052] FIGS. 1G and 2C illustrate the underside 320 of reagent slide 100. As shown in FIG. 2C, the bottom face 320 comprises struts 300, which provide stability and flexibility.
[0053] Figure 1H illustrates a sectional view of a pipette tip 360 contained in one of the pipette sheaths 120.
[0054] Although the Figures attached to this document illustrate a slide configured so that the one or more pipette sheaths, the one or more receptacles and the respective openings of the process tube and reagent tubes are all arranged linearly in relation to each other (that is, with midpoints on the same axis), those skilled in the art will realize that the supports disclosed in this document are not limited to specific configurations of receptacles, waste chambers, process tubes, pipette sheaths and reagent tubes. For example, some modalities propose a shorter reagent layer, for example, with staggered openings, in which some reagent, process tube or pipette tip openings occupy positions "offset from the axis". The various receptacles etc. they also do not need to occupy the same positions in relation to each other, as illustrated by FIGs. 1 to 3, in which the process tube is arranged approximately close to the support medium, liquid reagents are stored in receptacles mounted on one side of the process tube and receptacles containing solid reagents are mounted on the other side of the process tube. Therefore, in FIGs. from 1 to 3, the process tube is at one end of the slide and the pipette sheaths are at the other end, internally adjacent to a waste chamber and two or more reagent tubes. Still other arrangements are possible, such as arranging the process tube at one end of the holder, arranging the process tube adjacent to the pipette tips and pipette tip sheaths (as described in more detail in this document) and arranging the waste tube adjacent to the process tube. It will be realized that alternative configurations of the various parts of the reagent slide give rise only to variations in shape and can be framed in other variations of the apparatus as described, including, among others, sets of alternative instructions for the automated preparation and processing of samples.
[0055] Process tube 130 can also be a quick-fit tube, rather than part of a one-piece. Process tube 130 can be used in various mixing and reaction processes that take place during sample preparation. For example, cell lysis can occur in process tube 130, as well as nucleic acid extraction. The process tube 130 is then advantageously positioned in a location that, in general, minimizes operations that cause the movement of the pipette head involved in the transfer of liquids to the process tube 130.
[0056] Reagent tubes 140 are usually configured to contain multiple liquid reagents. For example, in some embodiments, the reagent slides may comprise three reagent tubes, in which the individual reagent tubes are provided with a sample wash buffer, a nucleic acid release buffer and a nucleic acid neutralization buffer, for example. example, to purify nucleic acids for NAT assays.
[0057] Reagent tubes 140 containing liquids or liquid reagents can be sealed by a laminated structure 400. The laminated structure can comprise a heat seal layer, a plastic layer, such as a polypropylene layer, and a metal layer, such as aluminum foil, where the heat seal layer is adjacent to one or more reagent tubes 140. The additional plastic film used in a laminate for receptacles with liquid reagents typically serves to prevent the liquid from coming into contact with aluminum. .
[0058] The exemplary modalities of a laminated structure 400, which differ in their layer structures, are described, for example, in the US Patent Application Publication 2009/0129978, incorporated by reference into this document. In some embodiments, the heat seal layer of laminated structure 400 is made, for example, of a lacquer or other polymer with a low melting point and is located on top of the reagent slide 100 when so applied, as shown in FIG . 5A. The laminated structure 400 may include a plastic layer 420 on top of the thermal seal layer 410, which is made of polypropylene and whose thickness is in the range of 10 to 50 microns. The laminated structure 400 may also include a layer of metal on top of the plastic layer, which comprises a layer of aluminum foil 440 bonded to the plastic layer 420 by an adhesive layer 430. Alternatively, the metal layer may be a layer of evaporated or sprayed metal in place directly onto the plastic layer, as shown in FIG. 5B.
[0059] The laminates implemented in this document facilitate long-term storage because the support includes the presence of sealed lyophilized reagents, as well as sealed liquids in close proximity, which is normally difficult to obtain.
[0060] In one embodiment, the top of the reagent tubes has beveled edges, such that when an aluminum sheet is thermally bonded to the top, the molten plastic does not extend beyond the outer edge of the tube. This is advantageous because, if the molten plastic decreases the inner diameter of the tube, it will cause interference to the pipette tip during operation. In other embodiments, a flat protruding part 260 facilitates the application and removal of laminate 400. The protruding surface 260, which is arranged on the upper face of the connecting member and which surrounds the inlet openings of the reagent tubes and, as an option, the waste chamber, is an optional feature of the support.
[0061] The way in which the liquid is pipetted out is such that a pipette tip piercing through the sheet will tear without creating a seal around the pipette tip. Sealing around the tip during pipetting would be disadvantageous because a certain amount of air flow is desirable for the pipetting operation. In this case, a seal is not formed because the laminated structure 400 causes the perforated sheet to remain in the position initially adopted when perforated. The top five frames in FIG. 4 illustrate pipetting a reagent out of a reagent tube with a laminate, as described in more detail in this document. In A, the pipette tip is positioned approximately in the center on the reagent tube 140, which contains reagent 270. In B, the pipette tip is lowered, normally in a controlled manner, to the reagent tube and, in doing so it, perforate sheet 280. The exploded view of this area illustrates the edge of the perforated laminate in contact with the pipette tip in the widest region where it penetrates the reagent tube. In C, the pipette tip is removed gradually, keeping the tip within the reagent concentration 270. The exploded view illustrates that the perforated sheet maintained the configuration it adopted when it was drilled and the pipette tip went down to its lowest position. deep inside the reagent tube. In D, the pipette tip absorbs reagent 270, possibly changing its height inside it. In E, the pipette tip is removed entirely from the reagent tube.
[0062] The materials of the various tubes and chambers can be configured to have at least a uniform internal surface and an internal surface coating that decreases the binding of nucleic acids and other macromolecules to it. The binding of nucleic acids is undesirable because of the possibility of causing less sensitivity in the subsequent detection and analysis of nucleic acids that are not trapped on the surface of the support. The process tube can also have a low bond surface and allow magnetic beads to run up and down inside the wall easily, without sticking to it. In addition, it has a hydrophobic surface coating, which allows static friction of fluids and, therefore, low binding of nucleic acids and other molecules. The reagent slides disclosed in this document can be made of many different polymers, including all thermoplastics, some thermosets and elastomers. Preferably, the material is suitable for injection molding. Non-limiting examples of polymers useful in making the sheets disclosed in this document include, for example, epoxy and phenolic polymers, nylon, polyethylene and polystyrene polymers, and the like. Preferably, the reagent slides are made of a plastic, such as polypropylene, and are of rigid dimensions, such that the reagent slides do not significantly lose their firmness or flex significantly under their own weight or deform easily during the process. routine handling and transportation and therefore do not allow leakage of reagents from them.
[0063] In addition, it should be considered consistent with the description revealed in this document that a support is also configured to receive a sample, such as in a sample tube. Thus, in embodiments described elsewhere, a rack receives a series of sample tubes and a series of corresponding supports in such a way that the sample tubes and supports are arranged separately and independently of each other. However, in other embodiments, a holder can be configured to also receive a sample, for example, in a sample tube. Thus, a complementary rack is configured to accept a series of supports, in which each support has a sample, in addition to reagents and other items. In this modality, the support is configured so that the sample is accessible to a sample identification tester. Kits
[0064] The reagent slides described in this document can be supplied in the form of a kit. For example, individual reagent slides can be packed together or individually in a sealed pouch to reduce the risk that air and moisture will come in contact with the reagents in the holder. This sealed pouch may contain one or more of the supports described in this document, such as 2, 4, 6, 8, 10, 12, 16, 20 or 24 supports.
[0065] The holder can also be provided as part of a sample preparation kit, in which the kit comprises a first pouch, which contains one or more of the supports described in this document, each of the supports being configured with liquid reagents for, for example, lysis, washing and release, and a second bag, which contains an inert atmosphere within it, and one or more reagent tubes containing lyophilized PCR reagents. This kit can also be configured to allow analysis of multiple samples and contain sufficient PCR reagents (or other amplification reagents, such as for RT-PCR, transcription-mediated amplification, tape shift amplification, NASBA, helicase dependent amplification , other reagents familiar to those skilled in the art and other reagents described in this document) to process those samples and a series of individual supports, such as 2, 4, 6, 8, 10, 12, 16, 20 or 24 supports.

权利要求:
Claims (29)
[0001]
1. Unified Reagent Slide, characterized in that it comprises: a slide with an upper face and a lower face, which comprises: a first and a second pipette sheaths comprising opposite sides, said first and second pipette sheaths comprising a first and a second pipette tip openings, respectively, each of which consists of a distinct opening in the upper face of the slide, in which the first and second pipette tip openings are configured for the introduction of the first and second pipette tips into the first and second pipette sheaths, respectively, and in which each of the first and second pipette sheaths is configured to surround the length of the first and second pipette tips, respectively, in which the first pipette sheath comprises a first core hole , the first orifice with core extending through a side wall of the first pipette sheath, in which the first orifice with core is configured to determine the presence, absence or length of a pipette tip within the first pipette sheath; a process tube; and a receptacle, comprising an opening through the reagent slide, wherein said receptacle is configured to receive a reagent tube.
[0002]
2. Unified Reagent Slide according to Claim 1, characterized in that the first pipette sheath comprises a pair of openings, the pair of openings comprising the first core hole and a second core hole extending through the wall side of the first pipette sheath, where the first and second core holes are located on opposite sides and are positioned the same distance along the length of the first pipette sheath with respect to the first pipette tip opening.
[0003]
3. Unified Reagent Slide, according to Claim 1, characterized in that the first and second pipette tip openings are located in adjacent positions along the upper face of the slide.
[0004]
4. Unified Reagent Slide according to Claim 1, characterized in that the process tube is integrated with the slide, wherein the slide comprises an opening of the process tube that forms an entrance to the integral process tube.
[0005]
5. Unified Reagent Slide, according to Claim 1, characterized in that it further comprises an integrated reagent tube, wherein said slide comprises an opening of the reagent tube that forms an entrance to the integrated reagent tube.
[0006]
6. Unified Reagent Slide according to Claim 1, characterized in that the first pipette sheath comprises a plurality of core holes.
[0007]
7. Unified Reagent Slide according to Claim 1, characterized in that the first pipette sheath comprises a plurality of pairs of openings.
[0008]
8. Unified Reagent Slide according to Claim 1, characterized in that the second pipette sheath comprises a first core hole, which extends through a side wall of the second pipette sheath.
[0009]
9. Unified Reagent Blade according to Claim 2, characterized in that the second pipette sheath comprises a second pair of openings, the second pair of openings comprising a third and fourth core holes extending through the side wall the second pipette sheath, where the first and second core holes are located on opposite sides and are at the same distance along the length of the second pipette sheath from the tip opening of the second pipette.
[0010]
10. Unified Reagent Slide according to Claim 1, characterized in that the first and second pipette sheaths are of different lengths extending from the tip openings of the first and second pipettes, respectively.
[0011]
11. Unified Reagent Slide according to Claim 1, characterized in that the sheath of the second pipette comprises a core hole.
[0012]
12. Unified Reagent Slide according to Claim 1, characterized in that the location of the first core hole is at a distance along the length of the first pipette sheath such that, when the tip of the first pipette is inserted in the sheath of the first pipette, the tip of the first pipette extends beyond the first orifice with core inside the sheath of the first pipette.
[0013]
13. Unified Reagent Slide according to Claim 5, characterized in that it comprises a plurality of integral reagent tubes.
[0014]
14. Unified Reagent Slide according to Claim 4, characterized in that it comprises a plurality of integral process tubes.
[0015]
15. Unified Reagent Slide according to Claim 1, characterized in that it comprises a plurality of reagent receptacles, wherein the plurality of receptacles is configured to receive a plurality of reagent tubes.
[0016]
16. Unified Reagent Slide according to Claim 1, characterized in that it comprises a waste chamber, which is integrated with the slide and in which the waste chamber comprises a waste opening that forms an entrance to the integral waste chamber.
[0017]
17. Unified Reagent Slide according to Claim 5, characterized in that the integral reagent tube comprises a liquid reagent.
[0018]
18. Unified Reagent Slide according to Claim 5, characterized in that it further comprises a laminate that seals the opening of the integral reagent tube.
[0019]
19. Method for Detecting the Presence or Absence of Pipette Tip Within Unified Reagent Slide Pipette Sheath, characterized in that the method comprises: providing the unified reagent slide as defined in Claim 3; providing an optical beam through the first core hole of the pair of openings; and detect whether the optical beam leaves unobstructed through the second orifice of the pair of openings, where the unobstructed exit of the optical beam through the second orifice of the first pipette sheath indicates the absence of the tip of the first pipette within the sheath of the first pipette. first pipette and where the obstructed outlet of the optical beam through the second orifice with core indicates the presence of the tip of the first pipette within the sheath of the first pipette.
[0020]
20. Method To Determine Pipette Tip Length Within Unified Reagent Slide Pipette Sheath, characterized in that the method comprises: providing a unified reagent slide, comprising: a slide with an upper face and a lower face, comprising: a process tube; a receptacle, comprising an opening through the reagent slide, wherein the receptacle is configured to receive a reagent tube; a first and a second pipette sheath, comprising: a tip opening of the first and second pipettes, respectively, each of which comprises a distinct opening in the upper face of the slide, in which the tip openings of the first and second pipettes are configured for introducing a tip of the first and second pipettes into the sheaths of the first and second pipettes, respectively, and in which each of the sheaths of the first and second pipettes is configured to surround the length of the tip of the first and second pipettes, respectively; a pair of upper openings in the pipette sheath and a pair of lower openings in the pipette sheath in the first pipette sheath, each of the pairs of upper and lower openings a first and a second core hole, extending through a side wall of the first pipette sheath, where the first and second orifices with the core of the pairs of upper and lower openings of the pipette sheath are located on opposite sides of the sheath of the first pipette and are located along the length of the sheath of the first pipette at the same distance from the tip opening of the first pipette and in which the pair of upper openings in the pipette sheath is located closer to the tip opening in the first pipette than the pair of lower openings in the pipette sheath; provide light through the first core hole in the pair of upper openings in the pipette sheath; provide light through the first core hole of the pair of lower openings in the pipette sheath; detect whether light is prevented from passing through the second orifice with the core of the pair of upper openings in the pipette sheath; and detecting whether light is prevented from passing through the second orifice of the lower core of the pair of lower openings in the pipette sheath, where the obstruction of light through the second orifice of the pair of upper openings in the pipette sheath and the passage of the light through the second core hole of the pair of lower openings in the pipette sheath indicates that the first pipette tip within the first pipette sheath has a length that does not extend to the pair of lower openings in the pipette sheath when inserted into the first pipette sheath.
[0021]
21. Method for Detecting the Presence or Absence of Pipette Tip Within Unified Reagent Slide Pipette Sheath, characterized in that it comprises: providing the unified reagent slide as defined in Claim 1; determine whether the tip of the first pipette extends into the pipette sheath from the tip opening of the first pipette at least as far as the first orifice with core.
[0022]
22. Method for Detecting the Presence or Absence of Pipette Tip Within Unified Reagent Slide Pipette Sheath, according to Claim 21, characterized in that the step of determining comprises visual inspection of the interior of the first pipette sheath.
[0023]
23. Method for Detecting the Presence or Absence of Pipette Tip Within a Unified Reagent Slide Pipette Sheath, according to Claim 21, characterized in that the determining step comprises: providing an optical beam that penetrates the sheath of the first pipette through the first core hole; and determining the reflection or obstruction of the optical beam as indicative of the presence or absence of the tip of the first pipette extending into the sheath of the first pipette by the distance from the tip opening of the first pipette to the first orifice with core.
[0024]
24. Method, characterized in that it comprises: providing a unified reagent slide, comprising: a slide with an upper face and a lower face, comprising: a process tube; a receptacle, comprising an opening through the reagent slide, in which the receptacle is configured to receive a reagent tube; a first and second pipette sheath, comprising: a tip opening of the first and second pipettes, respectively, each of which consists of a distinct opening in the upper face of the slide, in which the tip openings of the first and second pipettes are configured for inserting the tips of the first and second pipettes into the sheaths of the first and second pipettes, respectively, and where each of the sheaths of the first and second pipettes is configured to surround the length of the tips of the first and second pipettes, respectively, when present in the sheaths of the first and second pipettes; an orifice with an upper core and an orifice with a lower core within the sheath of the first pipette, each of the orifices with an upper and lower core crossing the side wall of the sheath of the first pipette, where the orifice with the upper core is located closest to the opening tip of the first pipette than the orifice with lower core; determining whether a pipette tip extends into the sheath of the first pipette from the tip opening of the first pipette to the first core hole; and determining whether a pipette tip extends into the sheath of the first pipette from the tip opening of the first pipette to the lower orifice with core.
[0025]
25. Method according to Claim 24, characterized in that the step of determining whether a pipette tip extends into said first pipette sheath from the opening of the first pipette tip to the upper core hole comprises visual inspection from the inside of the sheath of the first pipette through the hole with the upper core and the hole with the lower core.
[0026]
26. The method of Claim 24, wherein the step of determining whether a pipette tip extends into the first pipette sheath from the opening of the first pipette tip to the upper core hole comprises: providing a optical beam through the hole with upper core; and determining reflection or obstruction of the optical beam as indicative of the presence or absence of a pipette tip extending into the sheath of the first pipette from the tip opening of the first pipette to the orifice with the upper core.
[0027]
27. The method of Claim 24, wherein determining whether a pipette tip extends into the first pipette sheath from the opening of the first pipette tip to the orifice with a lower core comprises: providing an optical beam through the hole with a lower core; and determining the reflection or obstruction of the optical beam, as indicative of the presence or absence of a pipette tip extending into the first pipette sheath from the opening of the first pipette tip to the lower core orifice.
[0028]
28. The method of Claim 24, characterized in that the unified reagent slide further comprises a second core orifice and wherein the upper core orifice and the second core orifice are present on opposite sides and equidistant from the top the sheath of the first pipette.
[0029]
29. Method according to Claim 28, characterized in that determining whether a pipette tip extends into the sheath of the first pipette from the tip opening of the first pipette to the upper core orifice comprises: passing light through the upper nucleus; and detecting whether light passes through the second nucleus orifice.

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同族专利:

公开号 | 公开日

KR102121853B1|2020-06-12|

WO2013049706A1|2013-04-04|

AU2016201604A1|2016-04-07|

US10076754B2|2018-09-18|

EP3273253A1|2018-01-24|

BR112014007440A2|2017-04-04|

JP6117217B2|2017-04-19|

AU2012315595B2|2015-10-22|

JP2014528577A|2014-10-27|

EP2761305A1|2014-08-06|

CN103959070A|2014-07-30|

RU2014112980A|2015-11-10|

CA2849917C|2020-03-31|

EP3273253B1|2020-08-26|

US20170266666A1|2017-09-21|

US9480983B2|2016-11-01|

US20140206088A1|2014-07-24|

DK3273253T3|2020-10-12|

ES2825905T3|2021-05-17|

CN106996984B|2018-11-27|

RU2622432C2|2017-06-15|

CA2849917A1|2013-04-04|

AU2016201604B2|2017-11-16|

ES2645966T3|2017-12-11|

CN106996984A|2017-08-01|

JP2017129595A|2017-07-27|

CN103959070B|2017-05-10|

AU2012315595A1|2014-05-22|

AU2016200340A1|2016-02-18|

EP2761305B1|2017-08-16|

KR20140084105A|2014-07-04|

US20160107161A1|2016-04-21|

DK2761305T3|2017-11-20|

US9222954B2|2015-12-29|

JP6316473B2|2018-04-25|

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