Patent Application
20150299770
The present invention relates to the field of nucleic acid amplification, particularly to the use of a polymerase chain reaction to amplify nucleic acids. The invention provides methods and kits which can be used to amplify nucleic acids by combining an FTA™ Elute solid support with PCR reagents for one step amplification of nucleic acid samples. The invention has applications in the long term storage and easy processing of nucleic acids and is particularly useful in genotyping, diagnostics and forensics.
The polymerase chain reaction (PCR) is a common tool used in molecular biology for amplifying nucleic acids. U.S. Pat. No. 4,683,202 (Mullis, Cetus Corporation) describes a process for amplifying any desired specific nucleic acid sequence contained in a nucleic acid or mixture thereof.
Long-term storage, transport and archiving of nucleic acids on filter paper or chemically modified matrices is a well-known technique for preserving genetic material before the DNA or RNA is extracted and isolated in a form for use in genetic analysis such as PCR. Thus, EP1563091 (Smith et al, Whatman) relates to methods for storing nucleic acids from samples such as cells or cell lysates. The nucleic acid is isolated and stored for extended periods of time, at room temperature and humidity, on a wide variety of filters and other types of solid support or solid phase media. Moreover, the document describes methods for storing nucleic acid-containing samples on a wide range of solid support matrices in tubes, columns, or multiwell plates.
WO/9003959 (Burgoyne) describes a cellulose-based solid support for the storage of DNA, including blood DNA, comprising a solid matrix having a compound or composition which protects against degradation of DNA incorporated into or absorbed on the matrix. This document also discloses methods for storage of DNA using the solid medium, and for recovery of or in situ use of DNA.
U.S. Pat. No. 5,496,562 (Burgoyne) describes a cellulose-based solid medium and method for DNA storage. Method for storage and transport of DNA on the solid medium, as well as methods which involve either (a) the recovery of the DNA from the solid medium or (b) the use of the DNA in situ on the solid medium (for example, DNA sequence amplification by PCR) are disclosed. Unfortunately, the methods described only incorporates a surfactant or detergent on the surface of the solid medium and therefore suffer from the disadvantage that they require a separate step for the removal of the detergent before PCR is performed.
WO993900 (Gentra) describes again a method for processing and amplifying DNA. The method includes the steps of contacting the sample containing DNA to a solid support wherein a lysis reagent is bound to the solid support. The DNA is subsequently treated with a DNA purifying reagent and is purified. The application does not include a sequestrant on the solid support and requires a separate step for the removal of the lysis reagent and purification of the DNA before amplification.
WO9639813 (Burgoyne) describes a solid medium for storing a sample of genetic material and subsequent analysis; the solid medium comprising a protein denaturing agent and a chelating agent. The method described is for chelating agents which are any compound capable of complexing multivalent ions including Group II and Group Ill multivalent metal ions and transition metal ions. The invention does not specifically mention cyclodextrin as a chelating agent, nor does it suggest the PCR analysis could be performed in a single step.
U.S. Pat. No. 5,705,345 (Lundin et al.) describes a method of nucleic acid preparation whereby the sample containing cells is lysed to release nucleic acid and the sample is treated with cyclodextrin to neutralize the extractant. The advantage of this system is that conventional detergent removal requires a separation step however with the addition of cyclodextrin to neutralize the detergent it would remove the separation step needed and reduce chance of contamination.
GB2346370 (Cambridge Molecular Technologies Ltd) describes applying a sample comprising cells containing nucleic acid to a filter, the cells are retained by the filter and contaminants are not. The cells are lysed on the filter and retained alongside the nucleic acid. Subsequent steps filter out the cell lysate while retaining the nucleic acid.
WO9618731 (Deggerdal) describes a method of isolating nucleic acid whereby the sample is bound to a solid support and sample is contacted with a detergent and subsequent steps performed to isolate the nucleic acid.
WO0053807 (Smith, Whatman) describes a medium for the storage and lysis of samples containing genetic material which can be eluted and analysed. The medium is coated with a lysis reagent. In addition the medium could be coated with a weak base, a chelating agent, a surfactant and optionally uric acid.
WO9938962 (Health, Gentra Systems Inc.) describes a solid support with a bound lysis reagent. The lysis reagent can comprise of a detergent, a chelating agent, water and optionally an RNA digesting enzyme. The solid support does not contain cyclodextrin and requires further steps for purification of the nucleic acid for amplification analysis.
Current methods for DNA amplification involve a DNA purification procedure which often involves several steps which increases the chance of contamination. This is a tedious process and prior art methods have a number of clear disadvantages in terms of cost, complexity and in particular, user time. For example, column-based nucleic acid purification is a typical solid phase extraction method to purify nucleic acids. This method relies on the nucleic acid binding through adsorption to silica or other support depending on the pH and the salt content of the buffer. Examples of suitable buffers include Tris-EDTA (TE) buffer or Phosphate buffer (used in DNA microarray experiments due to the reactive amines). The purification of nucleic acids on such spin columns includes a number of complex and tedious steps. Nucleic acid purification on spin columns typically involves three time-consuming and complex steps/stages:
the sample containing nucleic acid is added to the column and the nucleic acid binds due to the lower pH (relative to the silanol groups on the column) and salt concentration of the binding solution, which may contain buffer, a denaturing agent (such as guanidine hydrochloride), Triton X-100, isopropanol and a pH indicator;
the column is washed with 5 mM KPO4 pH 8.0 or similar, 80% EtOH); and
the column is eluted with buffer or water.
Alternative methods involve the binding of nucleic acids in the presence of chaotropic salts such that DNA binds to silica or glass particles or glass beads. This property was used to purify nucleic acid using glass powder or silica beads under alkaline conditions. Typical chaotropic salts include guanidinium thiocyanate or guanidinium hydrochloride and recently glass beads have been substituted with glass containing minicolumns.
The best defence against PCR amplification failure in forensics applications is to combine sound sample handling and processing techniques with extraction systems proven to efficiently purify DNA.
Santos C. R. et al., (Brazilian Journal of Microbiology, 2012, 43, 389-392) describes a method of skipping the elution step prior to PCR amplification of nucleic acid and adding the punchers directly into the PCR mix. The PCR amplification was performed for the detection of HPV-DNA and was more efficient than the standard FTA elute card protocol of eluting the nucleic acid prior to amplification. However this method only used qualitative PCR to measure the presence of HPV.
Nozawa N. et al., (Journal of Clinical Microbiology, 2007, 45, 1305-1307) describes a method of using real time PCR for the detection of cytomegalovirus (CMV). The method described involved the use of filter paper with purified CMV and was added directly to the PCR mix. The paper noted that only instruments with a photo-multiplier-tube scanning system could be used for real time PCR assays with filter disks. The paper suggests that the filter paper would adversely affect instruments using a charge-coupled device camera and therefore teaches away from the use of filter papers in real time PCR machines such as an AB17700 machine.
Qiagen Sample & Assay Technologies Newsletter (March 2010, 15) describes the effects of a low A260/A230 ratio in RNA preparations on downstream PCR processing. The newsletter notes that increased absorbance at 230 nm in RNA samples is quite often due to contamination with guanidine thiocynate (a component of FTA elute cards and is used in RNA purification procedures). The experiments demonstrate A260/A230 ratio of an RNA sample is lower when guanidine thiocyanate is present, however guanidine thiocyanate concentrations up to 100 mM in an RNA sample did not affect the reliability of real-time PCR.
Typically the purification steps involved in the standard FTA elute card protocol can be cumbersome and purification can lead to a loss in DNA. There is therefore a need for an improved and simplified process for amplifying, quantifying and or profiling nucleic acid, which removes the need for a purification step. The present invention addresses this problem and provides methods and kits which can be used for single step amplification of nucleic acid from solid supports, particularly cellulose-derived supports.
The present invention provides methods and kits which can be used to amplify nucleic acids by contacting a solid support with nucleic acid and amplifying the nucleic acid in the presence of said solid support for easy amplification of DNA samples.
According to a first aspect of the present invention, there is provided a method for amplification of nucleic acid comprising the steps:
The advantage of amplifying the nucleic acid in the presence of the solid support is to reduce the number of steps required for nucleic acid amplification, thus saving operator time and facilitating operator usage.
In one aspect of the present invention, wherein the solid support is already in the reaction vessel prior to the addition of said cellular sample.
In another aspect, the method of amplification is a polymerase chain reaction.
In another aspect, the method of amplification comprises reverse transcription polymerase chain reaction, isothermal amplification or quantitative polymerase chain reaction.
In a further aspect, the nucleic acid amplification reagent solution comprises a polymerase, deoxyribonucleotide triphosphate (dNTP), a reaction buffer and at least one primer, wherein said primer is optionally labeled with a dye. Such dyes may include fluorescence dye FAM™ or CyDye DIGE Fluor™ from GE Healthcare (product code RPK0272). The nucleic acid amplification reagent solution can be present in a dried form, such as a “Ready-to-Go™” (RTG) format. The advantage of dried or lyophilised formulations of the polymerase chain reaction reagents is that they can be easily solublised by the addition of water, thus saving operator time and facilitating operator usage. To minimise operator error, the dried reagent mixture can be pre-dispensed into the reaction vessel, such as the well of a multi-well plate. Examples of such an RTG mixture include “Illustra Ready-to-Go RT-PCR beads” available from GE Healthcare (product code: 27-9266-01 Illustra Ready-To-Go RT-PCR Beads).
In a further aspect, the STR profile reagents are selected from the group consisting of PowerPlex 18D, PowerPlex 21, PowerPlex Fusion, Identifier Direct, Globalfiler Express and Y-Filer Direct.
In a further aspect, the nucleic acid is selected from the group consisting of DNA, RNA and oligonucleotide. The term “nucleic acid” is used herein synonymously with the term “nucleotides” and includes DNA, such as plasmid DNA and genomic DNA; RNA, such as mRNA, tRNA, sRNA and RNAi; and protein nucleic acid, PNA.
In one aspect, the chaotropic salt is a guanidine salt.
In another aspect, said guanidine salt is selected from the group consisting of guanidine thiocyanate, guanidine chloride and guanidine hydrochloride.
In one aspect, the chaotropic salt is sodium salt such as sodium iodide.
In another aspect, the solid support is washed with an aqueous solution following step i).
In one aspect, the solid support is selected from the group consisting of a glass or silica-based solid phase medium, a plastics-based solid phase medium, a cellulose-based solid phase medium, glass fiber, glass microfiber, silica gel, silica oxide, nitrocellulose, carboxymethylcellulose, polyester, polyamide, carbohydrate polymers, polypropylene, polytetraflurorethylene, polyvinylidinefluoride, wool and porous ceramics.
In another aspect, the solid support is a cellulose based matrix.
In a further aspect, said cellulose based matrix is in the form of a pre punched disc.
In another aspect, said cellulose based matrix is in the form of an FTA™ Elute card.
In another aspect, said cellulose based matrix is in the form of an indicating FTA™ Elute (iFTAe) Card wherein the dye indicates the presence of a biological sample.
In one aspect, wherein the amplified nucleic acid is quantified using a PCR imaging system.
In one aspect the cellular sample is selected from a group consisting of eukaryotic or prokaryotic cell, virus, bacteria, plant and tissue culture cells.
In another aspect, said cellular sample is selected from the group consisting of blood, serum, semen, cerebral spinal fluid, synovial fluid, lymphatic fluid, saliva, buccal, cervical cell, vaginal cell, urine, faeces, hair, skin and muscle. The cellular sample may originate from a mammal, bird, fish or plant or a cell culture thereof. Preferably the cellular sample is mammalian in origin, most preferably human in origin. The sample containing the nucleic acid may be derived from any source. This includes, for example, physiological/pathological body fluids (e.g. secretions, excretions, exudates) or cell suspensions of humans and animals; physiological/pathological liquids or cell suspensions of plants; liquid products, extracts or suspensions of bacteria, fungi, plasmids, viruses, prions, etc.; liquid extracts or homogenates of human or animal body tissues (e.g., bone, liver, kidney, etc.); media from DNA or RNA synthesis, mixtures of chemically or biochemically synthesized DNA or RNA; and any other source in which DNA or RNA is or can be in a liquid medium.
In a further aspect, the method is for use as a tool selected from the group consisting of a molecular diagnostics tool, a human identification tool, a forensics tool, STR profiling tool and DNA profiling.
In another aspect, wherein the nucleic acid is stored on the solid support prior to step ii).
In one aspect, the nucleic acid is stored on the solid support for at least 30 minute. The nucleic acid may be immobilised on the solid support for longer periods, for example, for at least 24 hours, for at least 7 days, for at least 30 days, for at least 90 days, for at least 180 days, for at least one year, and for at least 10 years. In this way the nucleic acid may be stored in a dried form which is suitable for subsequent analysis. Typically, samples are stored at temperatures from −200° C. to 40° C. In addition, stored samples may be optionally stored in dry or desiccated conditions or under inert atmospheres.
The method of the invention can be used either in single tube or a high-throughput 96-well format in combination with automated sample processing as described by Baron et al., (2011, Forensics Science International: Genetics Supplement Series, 93, e560-e561). This approach would involve a minimal number of steps and increase sample throughput. The risk of operator-induced error, such as cross-contamination is also reduced since this procedure requires fewer manipulations compared to protocols associated with currently used, more labour intensive kits (e.g. QIAmp DNA blood mini kit, Qiagen). The risk of sample mix-up is also reduced since the procedure requires few manipulations. Importantly, the method is readily transferable to a multi-well format for high-throughput screening. The present invention can thus improve sample processing for carrying out PCR reactions to aid genetic interrogations. The invention can be conducted in a 96 well/high throughput format to facilitate sample handling and thus eliminate batch processing of samples.
In a further aspect, the reaction vessel is a well in a multi-well plate. Multi-well plates are available in a variety of formats, including 6, 12, 24, 96, 384 wells (e.g. Corning 384 well multi-well plate, Sigma Aldrich).
In one aspect, the sample is transferred to the reaction vessel by punching or cutting a disc from the solid support. Punching the portion or disc from the solid support can be effected by use of a punch, such as a Harris Micro Punch (Whatman Inc.; Sigma Aldrich)
According to a second aspect of the present invention there is provided a method for amplification of nucleic acid comprising the steps:
In one aspect, wherein the solid support is already in the reaction vessel prior to the addition of said cellular sample.
In another aspect, wherein the method of amplification is a polymerase chain reaction.
In another aspect, said lysis reagent is selected from the group consisting of a surfactant, detergent and chaotropic salt.
In another aspect, the lysis reagent is selected from the group consisting of sodium dodecyl sulfate, guanidine thiocynate, guanidine chloride, guanidine hydrochloride and sodium iodide.
In a further aspect, said solid support is impregnated with sodium dodecyl sulfate (SDS), ethylenediaminetetracetic acid (EDTA) and uric acid.
In one aspect, said solid support is in the form of an FTA™ pre punched disc.
In another aspect, said cellulose based matrix is in the form of an indicating FTA™ (iFTA) Card wherein the dye indicates the presence of a biological sample.
In another aspect, the solid support is selected from the group consisting of a glass or silica-based solid phase medium, a plastics-based solid phase medium or a cellulose-based solid phase medium, glass fiber, glass microfiber, silica gel, silica oxide, nitrocellulose, carboxymethylcellulose, polyester, polyamide, carbohydrate polymers, polypropylene, polytetraflurorethylene, polyvinylidinefluoride, wool or porous ceramics.
In another aspect, the solid support is washed with an aqueous solution following step i).
In one aspect, wherein the amplified nucleic acid is quantified using a PCR imaging system.
In one aspect, wherein the cellular sample is selected from a group consisting of eukaryotic or prokaryotic cell, virus, bacteria, plant and tissue culture cells.
In another aspect, said cellular sample is selected from the group consisting of blood, serum, semen, cerebral spinal fluid, synovial fluid, lymphatic fluid, saliva, buccal, cervical and vaginal cells, urine, faeces, hair, skin and muscle. The cellular sample may originate from a mammal, bird, fish or plant or a cell culture thereof. Preferably the cellular sample is mammalian in origin, most preferably human in origin. The sample containing the nucleic acid may be derived from any source. This includes, for example, physiological/pathological body fluids (e.g. secretions, excretions, exudates) or cell suspensions of humans and animals; physiological/pathological liquids or cell suspensions of plants; liquid products, extracts or suspensions of bacteria, fungi, plasmids, viruses, prions, etc.; liquid extracts or homogenates of human or animal body tissues (e.g., bone, liver, kidney, etc.); media from DNA or RNA synthesis, mixtures of chemically or biochemically synthesized DNA or RNA; and any other source in which DNA or RNA is or can be in a liquid medium.
In a further aspect, the method is for use as a tool selected from the group consisting of a molecular diagnostics tool, a human identification tool, a forensics tool, STR profiling tool and DNA profiling.
In another aspect, wherein the nucleic acid is stored on the solid support prior to step ii).
In one aspect, the nucleic acid is stored on the solid support for at least 30 minute. The nucleic acid may be immobilised on the solid support for longer periods, for example, for at least 24 hours, for at least 7 days, for at least 30 days, for at least 90 days, for at least 180 days, for at least one year, and for at least 10 years. In this way the nucleic acid may be stored in a dried form which is suitable for subsequent analysis. Typically, samples are stored at temperatures from −200° C. to 40° C. In addition, stored samples may be optionally stored in dry or desiccated conditions or under inert atmospheres.
The method of the invention can be used either in single tube or a high-throughput 96-well format in combination with automated sample processing as described by Baron et al., (2011, Forensics Science International: Genetics Supplement Series, 93, e560-e561). This approach would involve a minimal number of steps and increase sample throughput. The risk of operator-induced error, such as cross-contamination is also reduced since this procedure requires fewer manipulations compared to protocols associated with currently used, more labour intensive kits (e.g. QIAmp DNA blood mini kit, Qiagen). The risk of sample mix-up is also reduced since the procedure requires few manipulations. Importantly, the method is readily transferable to a multi-well format for high-throughput screening. The present invention can thus improve sample processing for carrying out PCR reactions to aid genetic interrogations. The invention can be conducted in a 96 well/high throughput format to facilitate sample handling and thus eliminate batch processing of samples.
In a further aspect, the reaction vessel is a well in a multi-well plate. Multi-well plates are available in a variety of formats, including 6, 12, 24, 96, 384 wells (e.g. Corning 384 well multi-well plate, Sigma Aldrich).
In one aspect, the sample is transferred to the reaction vessel by punching or cutting a disc from the solid support. Punching the portion or disc from the solid support can be effected by use of a punch, such as a Harris Micro Punch (Whatman Inc.; Sigma Aldrich)
According to a third aspect of the present invention there is provided a kit for amplifying nucleic acid as herein before described and instructions for use thereof.
Chemicals and Materials Used
A list of the chemicals and their sources is given below:
Indicating FTA™ elute micro (WB120218 and WB120411);
Indicating FTA™ elute cassette (WB120230);
Normal human blood (Tissue Solutions Ltd);
Genomic DNA (Promega product code G152A);
Harris Uni-core punch, 1.2mm (Sigma, Catalogue number Z708860-25ea, lot 3110);
TaqMan Universal PCR master Mix, no AmpErase UNG (Applied Biosystems part number 4324018);
TaqMan RNase P Detection Reagents (Applied Biosystems part number 4316831)—contains RNase P primer;
PowerPlex 18D (Promega code DC1802)—contains primers;
Sterile water (Sigma Product code W4502);
Huma cervical epithelial cells (HeLa) (ATCC code CCL-2) and
Hi-Di Formamide (ABI code 4311320)
Experimental Results
STR Profiles from Hela Cell Spotted Elute FTA Cards
Cultured HeLa cells at a concentration of 2.5×106 cell/ml were spotted onto an indicating FTA elute (iFTAe) card. A 1.2 mm punch was taken from the cell spotted FTA elute card and combined with a direct STR kit PowerPlex 18D reaction mix for a final volume of 25 μl. The 25 μl sample mix was added to each well of a 96 well PCR plate prior to amplification. Samples were analysed on a 3130xl Capillary Electrophoresis using a 10 second sample injection.
PCR reaction was set up as follows:
Standards and samples were added to the appropriate wells. The plates were sealed and centrifuged at 1000 rpm for 1 minute. PCR was carried out on a Geneamp/ABI 9700 Thermo Cycler under the following thermal cycling conditions:
96° C. for 2min, followed by 28 cycles of: 94° C. for 10 sec, 60° C. for 1 min, followed by 60° C. for 20min, followed by a 4° C. Following amplification, visualisation of PCR products was achieved using Capillary Electrophoresis. The results are presented graphically in
Quantification of DNA from HeLa Cell Spotted and Blood Spotted FTA Elute Cards Using qPCR (Table 5).
Cultured HeLa cells at a concentration of 1×107 cell/ml or whole blood was spotted onto an indicating FTA elute card. A 3 mm or 1.2 mm punch was taken from the cell spotted FTA elute card and eluted using the iFTAe high throughput elution protocol or washed with 1 ml of elution buffer or left unwashed. Either the sample and FTA card or 5 μl of the eluate was added to the qPCR reaction containing TaqMan Rnase P detection reagents and TaqMan Universal PCR master mix. The PCR sample mix was added to individual wells of a 96 well PCR plate prior to amplification.
Indicating FTA elute high throughput elution protocol was as follows:
3 mm punch was added into a 96 well PCR plate, 200 μl of sterile water was added to each well, the plate was sealed and pulse vortexed three times (5 seconds each). The plate was centrifuged at 1200 rpm for 2 min. The water was aspirated and discarded and 60 μl of sterile water was added to each well and the plate was sealed again. The plate was centrifuged at 1200 rpm for 2 min and placed on a thermal cycler at 98° C. for 30 min.
The plate was then pulse vortexed 60 times (one pulse/sec) using a vortex mixer set on maximum speed. The plate was centrifuged at 1200 rpm for 2 mins and the eluate was removed from the wells using a pipette and transferred to another plate/well for quantification.
PCR reaction was set up as follows:
Standards and samples were added to the appropriate wells. The plates were sealed and centrifuged at 1000 rpm for 1 minute. PCR was carried out using Applied Biosystems 7900 Real-Time PCR System under the following thermal cycling conditions:
50° C. for 2 min, followed by 95° C. for 10 min, followed by 40 cycles of: 95° C. for 15 sec, 60° C. for 1 min. The detector used was the FAM™ probe. The results are presented in Table 5.
Table 5 shows the qPCR results of washed and unwashed blood spotted or cell spotted iFTAe card. The table shows the average yield of DNA from three qPCR reactions in ng/μl. The first 3 samples are replicates of DNA eluted from two 3 mm punches of iFTAe cards and the 4th sample is of a 1.2 mm punch of blood spotted iFTAe card that was washed with 1 ml of elution buffer and then amplified using real-time PCR (the data is the average of 3 separate samples). Samples 5 to 7 are replicates of DNA eluted from two 3 mm punch of an iFTAe card spotted with HeLa cells and the 8th sample is of a 3 mm punch HeLa spotted iFTAe card that was washed with 1 ml of elution buffer and then used in the real-time PCR machine. The last sample was of a 1.2 mm punch of HeLa spotted iFTAe card that was not washed and used directly in the real-time PCR machine (the data is the average of 3 separate samples). An unspotted negative punch did not yield any detectable DNA.
Quantification of DNA from HeLa Cell Spotted and Blood Spotted FTA Elute Cards Using qPCR (
Cultured HeLa cells at a concentration of 7.54×106 cell/ml or whole blood was spotted onto an iFTAe card. A 1.2 mm (HeLa and blood samples) punch was taken from the iFTAe card and washed with 1 ml of sterile water, vortexed and water was removed or the sample was left unwashed. A 3 mm (HeLa and blood samples) punch was taken from the iFTAe card and eluted using the iFTAe high throughput elution protocol. Either the sample spotted iFTAe card or 2 or 5 μl of the eluate was added to the qPCR reaction containing TaqMan Rnase P detection reagents and TaqMan Universal PCR master mix. The PCR sample mix was added to individual wells of a 96 well PCR plate prior to amplification.
Indicating FTA elute high throughput elution protocol was as follows:
For each sample to be processed 1×3 mm punch (HeLa sample) was placed into a 1.5 ml tube. 1 ml of sterile water was added to the tube and pulse vortexed three times (5 seconds each). The water was aspirated and discarded and the punches were transferred to the well of a 96 well PCR plate. 60 μl of sterile water was added to each well and the plate was sealed. The plate was centrifuged at 1200 rpm for 2 min and placed on a thermal cycler at 98° C. for 30 min. The plate was then pulse vortexed 60 times (one pulse/sec) using a vortex mixer set on maximum speed. The plate was centrifuged at 1200 rpm for 2 mins and the eluate was removed from the wells using a pipette and transferred to another plate/well for quantification. The plate was stored at 4° C. until quantification.
PCR reaction was set up as described above using Applied Biosystems 7900 Real-Time PCR System.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1220240.4 | Nov 2012 | GB | national |
| 1301344.6 | Jan 2013 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2013/073189 | 11/6/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61789788 | Mar 2013 | US |
