Methods for preparing samples for nucleic acid amplification

Abstract

The present invention is in the field of sample preparation. In particular, it relates to methods for preparing samples prior to performing nucleic acid amplification.

Claims

1. A method for passing a liquid sample through a porous solid matrix, comprising the steps of sealing the liquid sample within a container which comprises a porous solid matrix as at least a part of the container and raising a temperature to increase pressure inside the container, thereby to cause the liquid sample to pass through the porous solid matrix, wherein the liquid sample includes a biological sample and a buffer.

2. The method of claim 1, wherein the container comprises two or more different solid porous matrices.

3. The method of claim 1, wherein the liquid sample comprises nucleic acids and inhibitors of nucleic acid amplification.

4. The method of claim 3, wherein the container comprises a porous solid matrix which binds nucleic acids more strongly than inhibitors of nucleic acid amplification.

5. The method of claim 3, wherein the container comprises a porous solid matrix which binds inhibitors of nucleic acid amplification more strongly than nucleic acids.

6. A method for purifying nucleic acids from a liquid sample which comprises nucleic acids and inhibitors of nucleic acid amplification, wherein the method comprises the steps of (a) contacting the liquid sample with a porous solid matrix which binds inhibitors of nucleic acid amplification more strongly than nucleic acids, wherein heat is applied to the porous solid matrix and the liquid sample; and (b) separating the liquid sample comprising unbound nucleic acids from the porous solid matrix, wherein the liquid sample is passed through the porous solid matrix by sealing the liquid sample within a container comprising the porous solid matrix and the heat that is applied raises a temperature to increase pressure inside the container, thereby to cause the liquid to pass through the porous solid matrix, and the liquid sample includes a buffer.

7. The method of claim 6, wherein heat is applied to the liquid sample in step (b).

8. The method of claim 1, wherein the container comprises a flow restrictor which is configured to reduce flow of liquid sample from the container through the porous solid matrix compared to a container which does not have the flow restrictor.

9. The method of claim 8, wherein the flow restrictor is a filter, a frit, or a valve.

10. The method of claim 8, wherein the flow restrictor is a layer of material which melts at a temperature between 45 C. and 110 C.

11. The method of claim 10 wherein the layer of material is a wax.

12. The method of claim 1, further comprising a step of lysing the sample.

13. An apparatus for purifying nucleic acids according to claim 3, comprising a) the container comprising the porous solid matrix as at least a part of the container and means for sealing the container, the matrix being capable of binding inhibitors of nucleic acid amplification more strongly than nucleic acids, and b) a heating element configured to heat the container to a temperature of up to 110 C.; wherein the apparatus is configured to pass liquid through the porous solid matrix by heat.

14. The apparatus of claim 13, wherein the apparatus further comprises a second container to receive liquid passed through the porous solid matrix.

15. The apparatus of claim 13, wherein the apparatus further comprises a vessel comprising reagents for nucleic acid amplification.

16. An apparatus for purifying nucleic acids according to claim 6, comprising a) a container comprising a porous solid matrix as at least a part of the container and means for sealing the container, the matrix being capable of binding inhibitors of nucleic acid amplification more strongly than nucleic acids, and b) a heating element configured to heat the container to a temperature of up to 110 C.; wherein the apparatus is configured to pass liquid through the porous solid matrix by heat.

17. The apparatus of claim 16, wherein the apparatus further comprises a second container to receive liquid passed through the porous solid matrix.

18. The apparatus of claim 16, wherein the apparatus further comprises a vessel comprising reagents for nucleic acid amplification.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1

(2) FIG. 1a shows the principle of adding a liquid sample to a container which contains another, sealed opening; sealing the opening to the container where the liquid was introduced; opening an exit to the container on the other side of some sort of filter which will resist the flow of liquid out of this first container; placing the first container inside a collection vessel which can collect eluate from the container; placing the container and the vessel into a heating block and subsequently having liquid transferred from the container to the vessel.

(3) FIG. 1b, as for 1a, but with the container containing a solid phase material that preferably binds to NAAT-inhibitors rather than nucleic acid. The nucleic acid solution is found in the vessel post Heat-Elution.

(4) FIG. 1c, as for 1b but the solid phase material and sample are mixed prior to being added to the container.

(5) FIG. 1d, as for 1a, but with the container containing a solid phase material that preferably binds to nucleic acids rather than NAAT-inhibitors. The nucleic acid will be immobilised on the solid phase material post Heat-Elution within the first vessel.

(6) FIG. 1e, as for 1d, but the solid phase material and sample are mixed prior to being added to the first vessel.

(7) FIG. 1f, as for 1b or 1c, but the eluted nucleic acid is subsequently concentrated using a different solid phase material which preferably binds to nucleic acid. In this case, the solid phase material consists of paramagnetic beads which can be sedimented from a sample using a magnet. Nucleic acids may be released from the material with a suitable elution buffer, else, the beads can be added directly to a NAAT-based assay.

(8) FIG. 2

(9) Data showing heat-elution between two vessels plus and minus a solid phase material in the first vessel as described in Example 1.

(10) FIG. 3

(11) Showing typical BART-LAMP outputs for a dilution series of target nucleic acid. Using the BART technology, positive samples give an increase and then decrease in light intensity over time. The time to reach the light peak is inversely proportional to the amount of the targeted nucleic acid present in the amplification reaction.

(12) FIG. 4

(13) Comparison of Heated and Unheated IM-SDVB for inhibitor removal from ISO chocolate enrichment.

(14) FIG. 5

(15) Use of 4 SDVB and 3 SDVB to remove NAAT-inhibition from Coffee Enrichment. Compared to using no resin or the resin IM-SDVB (a), the resins 4 SDVB (b) and 3 SDVB (c) are shown to be extremely effective at removing inhibition caused by instant coffee.

(16) FIG. 6

(17) Humic acid inhibition removal by the resin mixture with heating time on the hot block at 100 C. (a); Temperature profile with time of the heated resin mixture on the hot block at 100 C. (b).

(18) FIG. 7

(19) Temperature profile with time of the Heat-Elution eluate from a hot block set at 100 C.

(20) FIG. 8

(21) Removal of Xylan inhibition by Heat-Elution vs. dilution in buffer only.

(22) FIG. 9

(23) Modulation of elution times by use of differing Frits and the use of wax.

(24) FIG. 10

(25) Amplification profile for C. difficile LAMP-BART reactions showing comparison between a C. difficile positive faecal sample extracted by A) Heat-Elution of a faecal IM SDVB-buffer mixture through a PVPP column, B) Spun elution of a PVPP column loaded with boiled faecal IM SDVB-buffer mixture and C) Spin clarification of a boiled faecal IM SDVB-buffer mixture.

(26) FIG. 11

(27) Intensity profile of UV visualised intercalator stained 1% agarose showing a Heat-Elution faecal extract and a centrifugally eluted faecal extract.

(28) FIG. 12

(29) Amplification profile for C. difficile LAMP-BART reactions showing following extraction of a C. difficile positive faecal sample and a C. difficile negative sample using Heat-Elution of a proprietary resin mix in a Pierce 8 ml column and a collection tube.

(30) FIG. 13

(31) (a) C. difficile LAMP BART detection from C. difficile positive clinical stool samples by Heat-Elution against dilutive methods;

(32) (b) Inhibitor Control LAMP BART profile from C. difficile positive clinical stool samples by Heat-Elution against dilutive methods

(33) FIG. 14

(34) Comparison of C difficile genomic DNA detection pre and post concentration by a simplified Charge Switch magnetic bead method.

(35) FIG. 15

(36) Integration of multi-vessel sample preparation & amplification using heat-elution

(37) FIG. 16

(38) Comparison of C difficile genomic DNA detection with and without performing Heat-Elution with Charge Switch magnetic beads incorporated into the first vessel.

EXAMPLES

Example 1: Heat is Sufficient to Drive Eluate from a Container Against a Resistive Force Using Standard Plastic Consumables and Solid-Phase Matrices Demonstrating the Heat-Elution Principle

(39) The heat within the sample preparation column permits a build up of pressure that allows self elution of the sample lysate through the column base, and does not require assistance of a centrifuge or syringe to achieve this (FIG. 1). This feature is exemplified on a (a) small 0.8 ml column scale as well as a (b) larger 8 ml column.

(40) (a) A 0.8 ml column (Pierce #89688) was filled with a proprietary resin mixture in reaction buffer to a final volume of 600 l where the excluded buffer volume was 327.5 l. The cap was closed tightly and the twist tab was broken off. The column was placed into a 2 ml collection tube and the entire column and tube was placed onto a heating block at 95 C. for 10 min. During this time pressure had built up within the column and the majority of the liquid was gradually driven out through the base of the column into the collection tube.

(41) (b) An 8 ml column (Pierce #89897) was filled with a proprietary resin mixture in reaction buffer to a final volume of 3.4 ml where the excluded buffer volume was 1.96 ml. The cap was closed tightly and the twist tab was broken off. The column was placed into a 13.5 mm internal diameter collection tube (Fisher #FB51579) and the entire column and tube was placed onto a bespoke heating block that has an insert depth of 80 mm. This was heated at 100 C. for 10 min. During this time pressure had built up within the column and the entire eluate was gradually driven out through the base of the column into the collection tube.

(42) To confirm that moderate heat can elute liquid phase via the Heat-Elution method in the present of a solid phase and solid-phase filter which creates back-pressure resisting elution a solution was eluted in the presence of Chelex 100 (Bio-Rad). Specifically, A 10% suspension of Chelex 100 in molecular grade water was made as follows: 1.096 g of Chelex was place in a 50 ml beaker and 10.96 ml of molecular grade water (Sigma) added. This was stirred on a magnetic stirrer following addition of a small flea. 800 l was added to two pre-weighed Pierce 89868 columns with their snap tabs already removed. These columns were placed in a 2 ml collection tube and centrifuged at 8000 rpm for 1 minute to remove the water from the Chelex resin in the column. 250 l of molecular grade water was added to these two columns and two other pre-weighed columns. These were placed into pre-weighed 2 ml snap cap tubes and placed on a 100 C. heat block or kept at room temperature for 5 minutes. At the end of the 5 minutes columns and eluate tubes were weighed to determine the volume of eluate and the volume of water left on the column. Neither of the columns held only at room temperature eluted any water, whereas, both the heated columns had eluted with 198.5 l eluted from the heated Chelex column and 226.4 l from the water only column (FIG. 2).

Example 2: The BART Reporter System

(43) The BART reporter system has been explained in detail in WO2004/062338 and WO2006/010948, which are hereby incorporated by reference. BART is an example of a reporter system designed for isothermal NAATs which gives a single type of signal from a sample, a bioluminescent signal. BART utilises the firefly luciferase-dependent detection of inorganic pyrophosphate. This is produced in large quantities when target sequences are detected using a NAAT. As such, molecular diagnostics can be achieved with BART simply by measuring the light emitted from closed tubes, in a homogeneous phase assay (FIG. 3). BART is proven with several different NAATs, operating between 50-63 C. The BART reporter is a particularly effective means to follow the rate of amplification of a NAAT since the light output represents a measure of the instantaneous rate of amplification (whereas e.g. fluorescent outputs show the accumulation of a signal and hence the measurements have to be differentiated to obtain the amplification rates).

Example 3: Solid Phase Materials which Remove NAAT Inhibitors Perform Better when they are Eluted at Higher Temperature (which Naturally Happens with Heat-Elution)

(44) i) Ability of the Solid Phase Resin 3 SDVB to Remove NAAT-Inhibitors at Different Temperatures

(45) 20 l of a 187.5 ng/l humic acid stock was added to BART-LAMP reaction buffer (190 mM Bicine, pH8.0) in two sets containing either 580 l of buffer with no 3 SDVB resin or 580 l of buffer with 3 SDVB at 20%. One set was vortex mixed, then heated at 95 C. for 5 minutes followed by a second vortex. The control set was vortexed and left at room temperature for this time then vortexed again. 20 l of each supernatant was used to reconstitute a freeze dried BART-LAMP reaction containing a fixed number (10.sup.4) of a particular target DNA molecule (referred to herein as the Inhibitor control). The peak times of duplicate reactions were subsequently compared. The presence of inhibitors may either slow or abolish amplification, in which case the peak times (the time it takes the BART reporter system to give the characteristic light peak) will increase or disappear altogether respectively. This showed that with no heat and no resin the reaction times with humic acid were 59.70 min. These improved to 57.00.5 min in the presence of resin and further significantly improved with the heated resin to 38.42.1 min.

(46) ii) Ability of the Solid Phase Resin IM-SDVB to Remove Complex NAAT-Inhibitors at Different Temperatures.

(47) The process of testing chocolate for food pathogens involves incubating 25 g of chocolate in 250 ml of an enrichment broth containing buffered peptone water with milk powder and the dye Brilliant Green. Following incubation it had been found that this enrichment broth was highly inhibitory to NAATs; this broths is referred to herein as ISO chocolate enrichment. The identity of the inhibitor(s) is unknown. Two sets of 20 l samples of an ISO chocolate enrichment were added to 580 l of 20 mM Tris buffer pH 8.8 containing 10 mM ammonium sulphate, 0.15% Triton X-100, 0.4 mg/ml polyvinylpyrrolidone and 0.09% sodium azide with 10% (w/v) IM-SDVB (a cation exchange resin consisting of a styrene divinylbenzene copolymer with iminodiacetate functionalised groups.) in 1.5 ml centrifuge tubes. These were pulse vortexed. One set of chocolate sample tubes were heated at 110 C. on a heating block for 5 minutes. The other set was kept at room temperature for the same time. After 5 minutes both sets of tubes were pulse vortexed, allowed to cool and 20 l from each tube used to reconstitute freeze dried inhibitor control BART-LAMP reactions in triplicate in 200 l PCR strips. FIG. 4 shows that the heated chocolate enrichment gave a mean peak time of 24.71.15 min, which is 8.1 min faster than the unheated mean peak time of 32.81.61 min. Blank inhibitor control ran simultaneously gave a peak time of 18.91.00 min. Therefore, heating the chocolate in IM-SDVB resulted in a reduction of inhibition of BART-LAMP compared to not heating.

(48) iii) Ability of the Solid Phase Resin PVPP to Remove the NAAT-Inhibitor Humic Acid at Different Temperatures.

(49) 200 l of PVPP suspension was pipetted to 200 l tubes that were spun down to give a dense PVPP bed and 100 l of the excess liquid removed. 100 l of 1.25 g/l humic acid was added to the top of the PVPP bed. The addition was vortex mixed throughout the PVPP. Three tubes were heated at 95 C. for 15 minutes and three tubes were left at room temperature for 15 minutes. All tubes were vortexed after 5 minutes and put back to temperature. 20 l was taken to a separate PCR tubes and particulates were spun down. Some of the room temperature humic acid over PVPP supernatant was also heated at 95 C. for 15 min. 5 l from each was added to 15 l Inhibitor Control BART-LAMP reactions Inhibitor control peak times for humic acid on PVPP heated at 95 C., humic acid on PVPP at room temperature and the supernatant from the latter heated at 95 C. were 32.857.20 mins, 47.638.17 mins and 59.1614.92 mins, respectively. This showed that heated PVPP removed more humic acid inhibitor than room temperature PVPP and that heating the humic acid supernatant from the room temperature PVPP extract gave no additional inhibition relief. In this study uninhibited peaks with water were at 23.113.31 mins.

(50) iv) Ability of the Solid Phase Resins 4 SDVB and 3 SDVB to Remove Complex NAAT-Inhibitors at Different Temperatures.

(51) Instant coffee can be demonstrated to contain potent NAAT inhibitors, therefore instant coffee represents a useful inhibitor model. Instant coffee (1 g) was added to 10 ml of buffered peptone water and incubated at 37 C. for 18 hours. 20 l of the enrichment was added to tubes of 580 l BART-LAMP amplification buffer containing no resin, 10% IM-SDVB, 300 l of 4 SDVB (a macroporous strong base anion exchange resin consisting of a styrene divinylbenzene matrix with quaternary amine functionalised groups) or 300 l of 3 SDVB (a macroporous styrene divinylbenzene copolymer with tertiary amine functionalised groups). All tubes were vortexed and heated at 110 C. for 5 min. Heated tubes were pulse vortexed and allowed to cool. 20 l was added in duplicate for each condition to reconstitute freeze dried inhibitor control BART-LAMP reactions in triplicate in 200 l PCR strips. Coffee enrichment in buffer without resin, with IM-SDVB (FIG. 5a), 4 SDVB (FIG. 5b) and 3 SDVB (FIG. 5c) gave an average peak times of 45.38.3 min, 37.83.8 min, 19.70.8 min and 22.41.5 min, respectively. Thus, both 4 SDVB and 3 SDVB have removed inhibitors from the coffee enrichment permitting inhibitor control peaking no more than 6 min slower than a water inhibitor control peak time of 17.10.0 min, compared to a 28 min delay for the coffee enrichment in buffer alone.

(52) v) Temperature Dependence of Inhibitor Removal

(53) Nine 2 ml tubes were filled with a particular resin mixture (10% v/v Chelex 100, 25% v/v Optipore SD-2 and 25% v/v Diaion WA30) in BART-LAMP reaction buffer where the excluded buffer volume was 633.2 Into each of these 21.8 l of a 187.5 ng/l stock of humic acid were added and vortex mixed. Each tube was placed on a heating block at 100 C. for time points 0, 1, 2, 3, 4, 5, 8 and 10 min, after which they were each removed from the heating block, then immediately vortexed and 200 l of the supernatant removed from the resins. The temperature in another tube was also monitored every 30 sec with a thermocouple throughout the 10 min heating. Two further controls were setup of 655 l buffer only (no inhibitor control) and 21.8 l humic acid in 633.3 l (inhibited control). These were heated on the hot block for 10 min. The supernatants from each were used to reconstitute freeze dried Inhibitor Control LAMP-BART reaction mixes in duplicate. These were run at 60 C. and the peak times of the reactions compared during the time course. Data in FIG. 6a showed that there was a trend of increased inhibitor removal with increased heating time, and with FIG. 6b this correlated to the temperature increase. By 5 min heating it was demonstrated that maximal inhibition relief was achieved, where the recorded temperature of the resin formulation reached 93.4 C. The inhibitor control peak time reduced from 43.20.5 min at 0 min incubation to 19.70.5 min after 10 min heating.

(54) A set of tubes with the proprietary resin mixture and humic acid was also prepared as unheated controls and incubated at room temperature. After time points 0, 1, 2, 3, 4, 5, 8 and 10 min, they were each immediately vortexed and 200 l of the supernatant removed from the resins. The supernatants from each were used to reconstitute freeze dried Inhibitor Control LAMP-BART reaction mixes in duplicate. These were run at 60 C. and the peak times of the reactions compared during the time course. The measured temperature of the resins by a thermocouple was 22.7 C. In these tubes the inhibitor control peak time at 0 min incubation was 49.01.0 min. Even after 10 min incubation the peak time was 44.22.7 min showing no significant reduction in inhibition with unheated resins, and confirming that heated resins were more effective in inhibitor removal.

(55) vi) Temperature Kinetics of Heat-Elution

(56) An 8 ml column (Pierce #89897) was filled with a particular resin mixture ((10% v/v Chelex 100, 25% v/v Optipore SD-2 and 25% v/v Diaion WA30) in BART-LAMP reaction buffer where the excluded buffer volume was 1.965 ml. The twist tab was broken off and the thermocouple was placed into the elution tip. The column was then placed into a 13.5 mm internal diameter collection tube (Fisher #FB51579) and the entire column and tube was placed onto a bespoke heating block that has an insert depth of 80 mm. This was heated at 100 C. and the temperature of the eluate monitored after 3 min, from when elution begins, every 30 sec for 10 min. It was confirmed that the optimal temperature correlating to maximal inhibitor removal (FIGS. 6a and 6b) was easily achieved during the heating time frame. FIG. 7 shows that an 8 min heating time allowed a rise in eluate temperature to >93 C. In fact since temperature is related to inhibitor removal then significant inhibitor removal would be occurring by 76 C. which translates into at least 3 min heating in this format.

(57) vii) Removal of Sample Eluate from Hot Resin Showed Better Inhibitor Removal than from Cold, in a Heat and Mixing Study.

(58) A 1 in 5 dilution of a an extract from a stool sample, which had been characterised as containing an abundance of NAAT inhibitors, was made in LAMP-BART reaction buffer. 50 l (10 mg) was mixed with 655 l buffer only as a no resin control. Further 50 l amounts were added to six tubes containing a proprietary resin cocktail of which the excluded volume of buffer was also 655 l. Each was heated at 95 C. for 10 min. Tubes were either (a) mixed both pre and post heating; (b) not mixed; (c) mixed before heating; (d) mixed after heating; (e) pre and post heat mixed and hot supernatant removed before cooling; (f) pre heat mixed and allowed to cool after heating then mixed. 20 l of each supernatant was used to reconstitute freeze dried Inhibitor control LAMP-BART reaction mixes in duplicate and run at 60 C. The peak times of the reactions were compared. Without resins, detection was not possible in the 120 min run time. With (b) and (c) there was also no detection indicating the initial mix with cold resin had no inhibitor binding effect. Reactions of (a), (d) and (e) all showed detection within 22.4 to 28.8 min where effective inhibitor removal occurred showing the immediate post heat mixing with hot resin was essential for inhibitor removal. If sample and resins were cooled to room temperature and mixed (f) then detection also failed within 120 min.

Example 4: Use of the Heat-Elution Method to Remove Inhibitors from Samples

(59) i) Heat-Elution can be Used to Remove the NAAT-Inhibitor Xylan

(60) 27.3 l of a 60 g/l Xylan stock was added to 655 l of reaction buffer and heated on a heating block at 100 C. for 6 min. 81.8 l of a 60 g/l Xylan stock was added to a column containing a proprietary resin mixture with an excluded volume of 1.965 ml. The cap was closed tightly and the twist tab was broken off. The column was placed into a 13.5 mm internal diameter collection tube (Fisher #FB51579) and the column and tube was placed into a conical flask of boiling water for 6 min.

(61) The eluates from each were used to reconstitute freeze dried Inhibitor control BART-LAMP reaction mixes in duplicate. These were run at 60 C. and the peak times of the reactions compared. FIG. 8 shows that without resin treatment the detection times were 39.52.1 min in the presence of xylan. However the column elutes gave detection times of 18.70.6 min indicating xylan inhibitor removal by the resins at the same dilution factor.

Example 5: Control of Elution

(62) i) Small Pore Sized Frits and High Melting Temperature Paraffin Wax Used to Modulate Elution

(63) For efficient sample inhibitor removal it was necessary that the sample was exposed to heated resins for a sufficiency of time before elution of the liquid phase. As such, it is necessary to control the rate of elution by some means.

(64) Elution rate was modulated by the use of a small pore sized polyethylene frit and a high melting temperature wax beneath the resins to constrict eluate flow out of the column. The start of elution during the heating was delayed by 3 min and complete before 10 min to ensure sufficient exposure of sample to heated resins, (FIG. 9).

Example 6: Quality of Eluted Nucleic Acids with Heat-Elution

(65) i) Heat-Elution can be Better than Centrifugation for Inhibitor Removal: Comparison Between Spun Elution and Heat Pressure Elution of a Faecal Extract by LAMP-BART

(66) 20% IM-SDVB in 27 mM dithiothreitol and 13.3 mM BICINE pH 5 was added to 250 l of a C. difficile positive diarrheal sample and vortex mixed. 200 l volumes of this vortex homogeneous mixture were added to an 800 l spin column containing a compact bed of PVPP or to two 1.5 ml tubes. The PVPP column containing the faecal-IM SDVB-DTT-BICINE mixture was capped tightly, the plastic tab at the bottom of the column removed to open the base of the column, placed in a 1.5 ml collection tube and heated on a hot block for 15 min at 105 C. (A). This resulted in eluate being Heat-Elution into the collection tube. The 1.5 ml tubes containing sample were simultaneously heated for 15 min at 105 C. When cooled, tubes were centrifuged at 14,000 rpm for 5 min and the supernatant from one of the tubes transferred to a bottom opened 800 l spin column containing a compact bed of PVPP in a 1.5 ml collection tube. This PVPP column was eluted by centrifugation at 8,000 rpm for 2 min on a microcentrifuge (B). The extract from the other 1.5 ml tube was used without PVPP column (C). 5 l volumes from each extract were added in duplicate to 15 l reaction volumes of C. difficile LAMP-BART reagents in the tubes of a PCR plate. These were covered with oil and placed on BART amplification detection instrument at 60 C. FIG. 10 shows the peak times for the faecal sample directly lysed in the PVPP column was 28.280.75 min, heat lysis in the tube and spinning on PVPP column was 28.282.26 min and heat lysing the faecal sample in buffer and spinning down the solids gave 61.393.78 min. Thus, for this particular faecal sample, heat lysing the sample and heat pressure elution within a PVPP column was as good as lysing the sample in a tube and then spin eluting the lysate on a PVPP column. The spun lysate gave slower detection due to the inhibitors still present in the lysate.

(67) ii) Heat-Elution Facilitates Elution of High Molecular Weight DNA

(68) A 0.8 ml column (Pierce #89688) was filled with a proprietary resin to a final dry volume of 555 l open for elution in a 1.5 ml tube. 333 l of a faecal sample was added to another tube containing 20% IM-SDVB in buffer and mixed by vortexing. 200 l of this mixture was added to the 0.8 ml column, tightly capped and then heat eluted sitting in its 1.5 ml tube on a 95 C. hot block for 15 minutes. Following elution, the tube was allowed to cool and the column transferred to a fresh tube and centrifuged at 8,000g for 3 min. 15 l of both heat and spun eluates were ran on a 1% agarose gel containing an intercalating stain after mixing with 3 l of loading buffer, and a gel image captured on a transilluminator (FIG. 11). The intensity profile of heat eluate shows that the stained DNA is substantially high molecular weight that remains in the well of the gel. Subsequent centrifugal elution of the same column shows additional low molecular weight staining. Centrifugation is known to cause shearing of high molecular weight DNA that results in low molecular weight fragments and can affect low copy number detection through the breakage within the target site for amplification. The absence of DNA shearing is advantage of heat pressure extraction elution.

Example 7: Demonstration of Heat-Elution Principle

(69) i) Use for Detection of C. difficile Following Extraction of Stool Samples

(70) 8 ml columns (Pierce #89897) were filled with a proprietary resin mixture in reaction buffer to a final volume of 3.4 ml where the excluded buffer volume was 1.965 ml. One sample each for C. difficile positive and negative faecal sample was sampled using a sterile micro ultrafine flocked swab (Puritan #25-3318 1PN 50). The end of the swab was mixed within the resin mixture, the stem of the swab snapped of and the column tightly closed. The twist tab was broken off and the column was placed into a 13.5 mm internal diameter collection tube (Fisher #FB51579) and the entire column and tube was placed onto a bespoke heating block that has an insert depth of 80 mm. This was heated at 100 C. for 10 min. During this time pressure had built up within the column and the entire eluate was gradually driven out through the base of the column into the collection tube. The eluates from each were used to reconstitute freeze dried Inhibitor control LAMP-BART reaction mixes in duplicate. These were run at 60 C. and the peak times of the reactions compared. This gave detection time for the C. difficile positive sample of 24.040.75 min whereas the C. difficile negative sample did not peak (FIG. 12), therefore showing that the method allows successful detection of C. difficile from stool.

(71) ii) The Heat-Elution Removes Faecal Inhibition without Compromising Detection by Avoiding the Need for Excessive Dilution.

(72) 68 ml columns (Pierce #89897) were filled with a proprietary resin mixture in reaction buffer where the excluded buffer volumes were 1.965 ml. Six confirmed C. difficile positive clinical stool samples were tested by the pressure column elution method. 150 l of a 1 in 5 dilution of each clinical sample in reaction buffer (30 mg) were added to each column and mixed. The twist tabs were broken off. The columns were then placed into a 13.5 mm internal diameter collection tubes (Fisher #FB51579) and the entire columns and tubes were placed onto a bespoke heating block that had an insert depth of 80 mm. These were heated at 100 C. for 10 min and the eluates cooled to room temperature. 20 l of the eluates were used to reconstitute freeze dried C. difficile and Inhibitor Control LAMP-BART reagent.

(73) The six stool samples were also diluted to levels in the order of those used in other available commercial C. difficile tests. These were prepared in reaction buffer to 1 in 200, 1 in 500 and 1 in 700 in final 600 l volumes, vortex mixed and heated in 2 ml tubes on a heating block set at 100 C. for 10 min. The tubes were mixed and then 20 l of the lysates were used to reconstitute freeze dried C. difficile and Inhibitor Control LAMP-BART reagent. Reactions were run at 60 C. for 90 min on the BART detection hardware. Samples A001 and A004 had a low C. difficile load, confirmed by high Ct values in the PCR method used by the Public Health Laboratory. Extraction by the Pressure Column had permitted detection of all replicates, including these two challenging samples where the dilutive methods between 200 and 700 dilution showed a compromise in detection. C. difficile LAMP-BART peak times for the methods are compared in FIG. 13a The Heat-Elution method required no more than 1 in 70 dilution in the resin mixture to sufficiently remove faecal inhibition.

(74) Sample A001 was a solid stool with a high inhibitor load and with the lowest C. difficile level in the set. This sample was successfully detected by the Heat-Elution method, where detection at 1 in 200 was compromised and 1 in 500 and 1 in 700 completely failed due to excessive dilution. FIG. 13b shows for this sample, the inhibitor control at 1 in 200 showed more inhibition than the eluate from the column (diluted to 1 in 70). In fact it was necessary to dilute the sample 1 in 500 to alleviate inhibition. In terms of inhibition removal, the Heat-Elution column with the resins was more effective than a 200-fold dilution as seen by the trends of the more inhibitory samples A001 and A005.

(75) iii) Detection of Norovirus Following Heat Pressure Extraction

(76) 50 l of a Norovirus GII-4 positive diarrheal sample was added to 150 l of 20% IM-SDVB in 20 mM MES, 40 mM DTT in a 1.5 ml screw cap tube. This was mixed, capped and placed on a 95 C. hot block for 10 min and then taken to ice for 2 min and then centrifuged at 17,000 g for 5 min. 100 l of the supernatant was added to a compacted PVPP bed in an 800 l tube and spun eluted at 8,000g for 2 min. 5 l volumes from the extract was added in duplicate to 15 l reaction volumes of norovirus GII-4 reverse transcriptase LAMP-BART reagent in the tubes of a PCR plate together with 1 l of the same faecal sample previously extracted using Boom technology. These were covered with oil and placed on BART amplification detection instrument at 60 C. The peak times for the faecal sample extracted by IM SDVB-MES-DTT heat lysis followed by PVPP column purification was 50.447.59 min. This compared to 43.460.76 min for 1 l from the previously Boom method extracted sample. This showed that Noroviral RNA could be extracted with a method that would be compatible with Heat-Elution.

Example 8: Concentration of Nucleic Acid from the Second Vessel Post Heat-Elution

(77) i) Post-Heat Elution, Nucleic Acids can be Further Concentrated.

(78) For low copy number applications, it was demonstrated that concentration of genomic C. difficile DNA levels were possible when spiked into reaction buffer i.e. the same reagent composition as the eluate.

(79) Dilutions of C. difficile genomic DNA were prepared to 10.sup.3, 10.sup.2, 10 and 0 copies per 20 l in reaction buffer. 20 l of each dilution was used to reconstitute freeze dried C. difficile LAMP-BART reaction mixes in duplicate and run at 60 C.

(80) Each dilution was also concentrated by taking 800 l of the spikes and mixing with 5 l of ChargeSwitch magnetic beads and 160 l of the kit binding buffer (Invitrogen) for 1 min. The supernatant was removed by settling the beads on a magnetic rack. The beads were re suspended in 80 l of reaction buffer and 20 l of the crude suspension, including beads were used to reconstitute freeze dried C. difficile LAMP-BART reaction mixes in duplicate and run at 60 C.

(81) FIG. 14 shows that concentration improved detection times of the 10.sup.3 and 10.sup.2 copies/20 l levels by 3.2 to 4.8 min, and permitted reproducibly in detection of both replicates at the 10 copies/20 l level before 52.3 min, where only 1 of 2 replicates were detected without concentration.

(82) ii) Concentration of C. difficile from Positive Faeces with a Simplified ChargeSwitch Magnetic Bead Method Post Heat-Elution

(83) C. difficile positive stool (1 in 5 in reaction buffer) was diluted 1 in 10 with a negative stool (prepared 1 in 5 in reaction buffer). 150 l (30 mg stool) of the dilution was applied to an 8 ml column (Pierce #89897) containing a particular resin mixture (10% v/v Chelex 100, 25% v/v Optipore SD-2 and 25% v/v Diaion WA30) in BART-LAMP reaction buffer and mixed by hand. The excluded buffer volume was 1.965 ml. The cap was closed tightly and the twist tab was broken off. The column was placed into a 13.5 mm internal diameter collection tube (Fisher #FB51579) and the entire column and tube was placed onto a bespoke heating block. This was heated at 100 C. for 10 min. 20 l of the collected eluate was used to reconstitute freeze dried C. difficile LAMP-BART reactions in duplicate.

(84) 800 l of the eluate was also concentrated by mixing with 5 l of ChargeSwitch magnetic beads and 160 l of the kit binding buffer (Invitrogen) for 1 min. The supernatant was removed by settling the beads on a magnetic rack. The beads were resuspended in 80 l of reaction buffer and 20 l of the crude suspension, including beads were used to reconstitute freeze dried C. difficile LAMP-BART reaction mixes in duplicate.

(85) The LAMP-BART reactions were run at 60 C. on the BART detection instrument. Concentration of the eluate improved detection time from 27.20.5 min (un-concentrated) to 19.20 min (post concentration).

(86) iii) Comparison of C difficile Genomic DNA Detection with and without Performing Heat-Elution with ChargeSwitch Magnetic Beads Incorporated into the First Container.

(87) 20 l of C. difficile genomic DNA (103 copies per 200 stock was added to 1.98 ml of Bicine buffer. 200 l of this was used to make serial dilutions with 1.8 ml BICINE buffer at 102 and 101 copies per 20 l. The gDNA dilutions were treated as follows:

(88) Set 1: No treatment. 20 l was used to directly reconstitute C difficile LAMP BART assays.

(89) Set 2: 400 l of C. difficile gDNA dilution was added to a 1.5 ml Heat-Elution column with a 2.7 mm frit with additional glass filter (G/FD FD #1823-025 paper, Whatman) with 5 l Charge Switch beads and 80 l of binding buffer. This was mixed by pipetting. The column lid was secured tightly and placed on the heating block (with a 2 ml collection tube) at 100 C. for 6 min. After elution, the GF/D material with the captured beads was transferred directly to 40 l of reconstituted C difficile LAMP BART assay

(90) The conditions with Heat-Elution and magnetic bead concentration helped to detect 1 log lower in dilution series as compared to no Heat-Elution, so demonstrating that Heat-Elution can be used in conjunction with bead-capture methods to concentrate nucleic acids in the Heat-Elution container (FIG. 16).

Example 9: Integration of Multi-Container Sample Preparation & Amplification Using Heat-Elution

(91) The principle of heat-elution can be applied such as to combine two or more associated containers which each perform a different function for sample preparation. A single heating block could be used to house such an association of containers, or a number of heating blocks could be used where the timing and rate of heating and final temperature of the heating block is designed to drive the sample in a coherent fashion through the containers.

(92) Further, one vessel could contain NAAT reagents such that the combination of containers and the vessel allows for direct addition of processed sample to NAAT reagents. As such, but appropriate design of heating blocks, once sample is added to the first container, the Heat-Elution method could perform all the steps of sample preparation and allow for adding sample to NAAT reagents and further allowing amplification to proceed (FIG. 15).

Example 10: Heat-Elution can Provide Samples for PCR

(93) Use of Lysate in PCR Detection

(94) An 8 ml column (Pierce #89897) was filled with a proprietary resin mixture in reaction buffer to a final volume of 3.4 ml where the excluded buffer volume was 1.965 ml. A C. difficile faecal sample was sampled using a sterile micro ultrafine flocked swab (Puritan #25-3318 1PN 50). The end of the swab was mixed within the resin mixture, the stem of the swab snapped off and the column tightly closed. The column was hand mixed and was placed onto a bespoke heating block that has an insert depth of 80 mm. This was heated at 100 C. for 10 min. The column was allowed to cool and lysate then removed from the top of the column. A C. difficile real time PCR reaction mix was prepared using the IQ Supermix (Bio-Rad #170 8862). 4.5 l of lysate was added to the PCR reactions in duplicate and ran on the ABI-PRISM 7000 together with a C. difficile genomic DNA dilution series and amplified following an initial denaturation step at 95 C. for 3 min by 50 cycles 94 C., 57 C. and 72 C. with each at 30 seconds. The lysate gave a Ct value of 29, which corresponded to a copy number of 1.1810.sup.4 when calculated from the calibration curve, indicating that the column lysate can also be used for real time PCR detection.