Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/505—Containers for the purpose of retaining a material to be analysed, e.g. test tubes flexible containers not provided for above
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers

Definitions

• This invention relates to nucleic acid material amplification and detection and is more particularly concerned with reaction cuvettes or devices, and methods, used to amplify and detect such materials.
• DNA detection is described in EP-A-0 381 501 using a method wherein polymerase chain reaction (PCR) amplification of miniscule amounts of nucleic acid material, and detection of the amplified material can all occur in a single cuvette which keeps the amplified material from escaping.
• PCR polymerase chain reaction
• Six temporarily-sealed blisters, also called compartments, are provided along with passageways connecting them to a detection site in a detection compartment.
• the blisters provide, in order, a PCR reaction compartment; a first wash compartment; an enzyme-labeling compartment containing, for example, streptavidin horseradish peroxidase (hereinafter SA-HRP); a second wash compartment; a compartment containing signalling material responsive to the enzyme; and a stop solution compartment.
• SA-HRP streptavidin horseradish peroxidase
• the Roche procedure incubates "at 37°C for 15 minutes with an avidin-horseradish peroxidase conjugate", which of course corresponds to the emptying of the enzyme blister of the cuvette described in EP-A-0 381 501 for the very same purpose. Thereafter, the Roche procedure" again washed the plate four times" "to remove unbound conjugate.” This, of course, corresponds to the second wash step provided by the second wash blister disposed between the enzyme blister and the signalling material blister in the cuvette of EP-A-0 381 501.
• EP-A-0 572 057 discloses a test pack and its method of use for the detection of amplified nucleic acid material.
• the method taught by EP-A-0 572 057 concerns hybridizing amplified nucleic acid materials to an immobilized probe situated within a "detection blister", through which reagents and fluids, housed in other discrete chambers (or blisters), are passed in an ordered sequence to produce a detectable signal.
• a wash chamber containing a wash solution.
• WO-A-9216659 discloses an integral "flow-through" test element (or device) and method for use thereof for the detection of a PCR-amplified target nucleic acid sequence.
• the test device of WO-A-9216659 comprises, inter alia, four reagent-containing blisters (or chambers) of which one blister is a wash chamber containing a wash solution.
• the device disclosed in WO-A-9216659 provides for a wash chamber and its method of use teaches a washing step between the steps of labelling and detection.
• non-specific signal that is, signal which occurs because of either the presence of unbound nucleic acid material which is not the target, and/or unbound SA-HRP which should not be present because the target nucleic acid material is not present.
• a method of detecting amplified nucleic acid material by hybridizing such material to a detection site comprising at least one immobilized probe, labeling the hybridized and now-immobilized nucleic acid material by bringing to the detection site a label which is or activates a signalling material to produce a signal, and thereafter adding the signalling material to the detection site to produce a detectable signal, characterized in that the labeling step is used directly after the hybridizing step without requiring a wash step in between, and the adding step is used directly after the labeling step without requiring a wash step in between.
• a device for amplifying and detecting nucleic acid material by using at least one target strand as a template comprising:-
• the invention is useful regardless of whether PCR amplification is used or not, and regardless of the presence of all the features of that cuvette, provided that no wash compartment is included with no intervening wash step as a result.
• wash or “wash solution” means, a solution substantially free of capture, label and signal-forming reagents used in the other compartments, that is, in either the label compartment or the signalling material compartment.
• each slug of liquid passes over the detection site(s), improving the efficiency.
• the optional shear-thinning gel which can be added as described hereinafter enhances this capability, in that it appears to create a more viscous slug which retards backward migration of the components that are removed by the slug's front boundary.
• FIG. 1 illustrates one form of the invention described in US-A-5 229 297, in which the wash compartment and wash step in between the reaction compartment and the label compartment has been eliminated.
• a reaction cuvette or device 10 comprises an inlet port 22 for injection of patient sample liquid, which connects via a passageway 21 to a PCR reaction compartment 26.
• a seal 46 temporarily blocks flow out of compartment 26. When seal 46 is broken, liquid feeds via a passageway 44 to a detection chamber 40 having sites 41 comprising, preferably, beads anchored in place which will complex with any targeted analyte passing them from compartment 26, and then with reagents coming from the other reagent compartments.
• Those other compartments are compartments 30, 32, 34, each feeding via passageways 48 and 50 to chamber 40. Each of those passageways is temporarily sealed at 56, and contains an appropriate reagent liquid.
• the wash compartment preferably comprises a buffer, surfactants, EDTA, NaCl, and other salts.
• Compartment 26 in addition to the patient sample added by the user, preferably includes all the conventional reagents needed for PCR amplification, optionally kept in place by temporary seal 25.
• the reagents can be pre-incorporated, or added with the patient sample as the latter is introduced.
• the reagents include primers which are bound to one member of a binding pair, the other member of which appears in compartment 30 described below.
• a useful example of the binding member attached to a primer is biotin. (If present, Seal 25 is burst by injecting sample.)
• Compartment 30 comprises, preferably, a label such as an enzyme bound to a complexing agent, such as avidin, which is a member of a binding pair, the other member of that pair being bound to a primer which becomes part of a targeted analyte during amplification in the reaction compartment 26 as described above.
• a useful reagent in compartment 30 is streptavidin horseradish peroxidase (hereinafter, SA-HRP). The other member of that binding pair is then biotin.
• Labels other than enzymes are also useful.
• fluorescent, radioactive, and chemiluminescent labels are also well-known for such uses.
• Chemiluminescent labels also preferably use a compartment 34 containing signalling reagent, discussed below for enzyme labels.
• Compartment 32 preferably comprises a wash solution as the reagent in Figure 1. However, in Figure 3 (an embodiment of the present invention) wash compartment 32 is absent.
• Compartment 34 preferably comprises signalling material, and any dye stabilizing agent which may be useful.
• a useful reagent solution in compartment 34 is a solution of a leuco dye which is a conventional substrate for the enzyme of compartment 30. H 2 O 2 and any shear-thinning gels are also included.
• Compartment 42 is a waste-collecting compartment, optionally containing an absorbant.
• Roller 60 exemplifies the exterior pressure means used to burst each of the compartments sequentially, to sequentially advance the contents of the respective compartment to detection chamber 40. Because all of the compartments and passageways remain sealed during the processing, no leakage out of the device occurs and carry-over contamination is prevented. Sealing of port 22 is achieved by folding corner 70 about fold line 72, so that hole 74 fits over port 22 and passageway 21 is pinched off. A closure cap is then used to keep corner 70 so folded.
• a useful processor to process device 10 is shown in EP-A-0 402 994.
• Such a processor uses a support surface on which devices 10 are placed in an array, and pressure members, for example, rollers, are mounted in position to process each of the cuvettes in parallel.
• the rollers are journalled several to one or more axles for convenience, these axles being incrementally advanced by gearing.
• the support surface is horizontal or tilted up to 15° from horizontal.
• heaters can be optionally included, either in stationary form or carried with the rollers.
• one and only one wash compartment 32 is used in Figure 1, to provide a wash step after incubation of the SA-HRP at the sites 41 of compartment 40, to remove any unbound SA-HRP.
• no wash step or wash liquid needs to be provided between the respective sequential movements of the amplified nucleic acid material and the SA-HRP, to sites 41, for the reason that each reagent directed to the detection site is effectively washed out by the next reagent entering the station. It is surprising that the small volume in each compartment is adequate to do this.
• the wash compartment can be supplemented, if desired, with additional wash liquid.
• Figure 2 is to add a wash compartment adjacent to the first wash compartment, so that initially the first wash compartment is emptied to the detection site, and then the second wash compartment. Parts similar to those previously described bear the same reference numeral, to which the distinguishing suffix "A" is appended.
• cuvette 10A involves the exact same features as in the comparative embodiment of Figure 1, except that an additional temporarily-sealed compartment 36 of wash liquid is interposed between compartments 32A and 34A. Passageway 52 connects it to compartment 40A, after seal 56A of compartment 36 is burst.
• a single wash compartment but with a greater volume of wash can be used.
• cuvette 10B comprises all the features of the previously described embodiments, except there is no wash compartment at all.
• the only compartments are the thermal cycling reaction compartment 26B, the label-containing compartment 30B (with, for example, streptavidin horseradish peroxidase), and compartment 34B containing the signalling material, for example, H 2 O 2 , optionally a shear-thinning gel described immediately hereafter, and a leuco dye which reacts with the label enzyme to produce a dye.
• an optional ingredient for inclusion with the signalling material is an approximate 0.5% agarose solution, to stabilize color formation at the detection sites in the detection compartment.
• Agarose has the shear thinning behavior that its viscosity at about this concentration drops 270mPas (27 poise) between a shear rate of 1 to 10 2 s -1 (more than 60% of its drop), and only another 30mPas (3 poise) for rates above 10 2 s -1 , when measured at 40°C.
• Other shear-thinning gels of similar viscosity behavior and low percentage concentration can also be used.
• an enlarged detection site 41B comprising immobilized beads as described in the aforesaid EP-A-0 381 051.
• the amplified target nucleic acid material with a biotin tail is shown as " ⁇ B ".
• SA-HRP is shown as "A*" as a labeled avidin.
• HUT/AAV/78 cells containing one copy of HIV per cell were treated in a standard phenol chloroform extraction process to isolate the DNA, and the amount of DNA obtained was quantified on a spectrophotometer.
• the recovered DNA (100,000 copies HIV) was amplified by polymerase chain reaction (PCR) in a cocktail containing each of the primers identified below (1 ⁇ M each), buffer [10mM magnesium chloride, 50mM tris(hydroxymethyl)aminomethane (TRIS), 50mM potassium chloride, and 0.1mg/ml gelatin], 1.5mM of each of dATP, dCTP, dGTP, and dTTP deoxynucleotide triphosphates, and 40 units of DNA polymerase obtained from Thermus aquaticus .
• PCR polymerase chain reaction
• Two sets of primers were used, one set complementary to the ENV region, and one set complementary to the GAG region of the HUT/HIV DNA, as is known to be used in multiplexing.
• One primer in each set was biotinylated to facilitate detection.
• PCR protocol was carried out using 250 ⁇ l of the above cocktail in the PCR reaction blisters of PCR analytical elements of the type described in EP-A-0 381 051 and US-A-5 299 297. More specifically, the cuvette 10C of Figure 7 was used. Parts similar to those previously described bear the same reference numeral with the letter “C” appended. Thus, compartments 26C, 30C, 32C, 36C and 34C; passageways 44C, 48C, 50C and 52C; detection site 40C, and waste compartment 42C were used as described above, except for the layout, or as noted hereinafter. For one thing, PCR amplification was done in a cuvette separate from the test cuvette 10C, with the amplified material being pooled and then injected into compartment 26C for consistency of results in all replicates, for example, 32 in Example 1.
• a thermal cycling processor of the type described in EP-A-0 402 994 was used.
• the target DNA was preheated to 90°C for 10s, then denatured at 96°C for 30s and cooled to 70°C for 60s to anneal primers and produce primer extension products. The latter two steps (heating at 96°C, then 70°C) were repeated for a total of 40 cycles.
• This PCR process was replicated 64 times, and the fluid containing the newly made PCR product was transferred from the 64 PCR blisters into a common vessel to create a pool of PCR product. Samples from this pool were diluted 1:20 in the PCR buffer described above for use in the tests described hereinafter.
• a wash solution was prepared to contain 1% sodium decyl sulfate in phosphate buffered saline solution containing 10mmolar sodium phosphate, 150mmolar sodium chloride, and 1mmolar ethylenediaminetetraacetic acid, pH 7.4.
• a conjugate of streptavidin and horseradish peroxidase obtained from Zymed Labs (San Francisco, CA) was diluted 1:8000 with casein (0.5%) in a phosphate buffer solution (pH 7.3) containing thimerosal preservative (0.01%).
• a solution of 25 g of polyvinylpyrrolidone in 100ml of water was mixed with a solution of 0.20g of 4,5-bis(4-dimethylaminophenyl)-2-(4-hydroxy-3,5-dimethoxyphenyl)imidazole blue-forming leuco dye in 1ml N,N-dimethylformamide and stirred for 1 hour.
• a poly[styrene-co-3-(p-vinylbenzylthio)propionic acid] (mole ratio 97.6:2.4, weight ratio 95:5, 1 ⁇ m average diameter) aqueous polymer particle dispersion was prepared, and an oligonucleotide described hereinafter was covalently bound to one portion of the polymer particles, and another oligonucleotide was covalently bound to another portion of the polymer particles using the procedures described in US-A-5 149 737 and in EP-A-0 462 644.
• the oligonucleotides were linked to the polymer particles through two tetraethylene glycol spacers, a 3-amino-1,2-propanediol moiety, and a thymine base. Each oligonucleotide was appended to the polymer particles through the amino group of the 3-amino-1,2-propanediol moiety to form reagents by the procedures of US-A-4 962 029.
• the polymer/oligonucleotide particle probes were mixed with a latex adhesive of poly(methyl acrylate-co-sodium 2-acrylamido-2-methylpropanesulfonate-co-2-acetoacetoxyethyl methacrylate) (90:4:6 weight ratio) at a dry weight ratio of particles to adhesive polymer of 4/0.1 (2.5% adhesive).
• the aqueous dispersion had a solids content of 4%.
• control reagent oligonucleotide sequence is a sequence from the HIV genome and was employed as a nonsense sequence. This nonsense probe should not capture any of the HUT/HIV analyte sequences, and consequently, no dye development should occur on the control reagents.
• the other probe reagent sequence was complementary to a sequence in the ENV region of the HUT/HIV DNA.
• the above reagents were used to prepare a series of analytical elements (cuvettes), each having reagent compartments (one of which is a PCR reaction blister into which the sample analyte is first introduced) a detection compartment, and a waste reservoir.
• the analytical devices were prepared by heating a sheet of poly(ethylene terephthalate)/polyethylene laminate (SCOTCHPAKTM 241, 3M Co.) at a forming station (or mold) to form an array of depressed areas (blisters) toward one side of the sheet, and a larger depressed area near the end, and at the other side of the sheet, to which a main channel ultimately leads, a main channel from the first blister to the last, and tributary channels from each blister to the main channel so that upon lamination to a cover sheet at a later time, the resulting cuvette had narrow channels leading from the depressed areas to a main channel analogous to the devices described in US-A-5 299 297.
• Each depressed area except the one at each end of the main channel was filled with an appropriate reagent composition.
• a cover sheet was laminated to form a cover over the depressed and channel areas, and sealed to create a burst seal between each depressed area (except the last one) and the channel leading from it to the main channel.
• the cover sheet was treated overall with corona discharge.
• the probe reagent formulations described above were then immediately deposited in four alternating spots on the treated surface, each spot having 0.9 to 1.1 ⁇ l of formulation noted hereinafter, in a row.
• the disposed formulations were dried for 30s in a stream of air at room temperature while heating the opposite side of the support with an iron at about 95°C.
• the cover sheet was then laminated and sealed in three steps.
• the sandwich was pressed and sealed by heating at about 149°C only around the blisters containing the reagent solutions and around the waste blister.
• the formation of the sample-receiving PCR blister, including burst seals, and the channels was completed by heating the test pack between appropriately shaped heating jaws at about 163°C.
• the third step was the formation of perimeter seals around the test pack, and resealing all blister perimeter seals using a top plate temperature of 199°C while the bottom plate remained at ambient temperature.
• the channels and blisters formed in the completed test pack (or element) were located so that passage of a roller across the portion of the element containing the reagent blisters would sequentially burst the seals of the blisters and force the reagent from each blister into and along an exit channel to the main channel leading to the area containing the capture probes.
• the finished element was inverted so that the cover sheet containing the capture probe spots (deposits) is the bottom of the finished element with the probe deposits properly aligned in the main channel to form a detection station.
• the four probe spots were located in different positions of the main channel in several samples.
• a last waste compartment located at the end of the main channel was larger than the others and fitted with an absorbent to be a reservoir for waste fluids, for both Example 1 and the Comparative Example.
• Example 1 The completed cuvettes of Example 1 and the Comparative Example were used to evaluate the reagent formulations as follows:
• a blister in each test device was filled (190-210 ⁇ l) with a 20X dilution of the PCR product described above and processed as follows:
• the analyte was preheated to 95°C for 120s and its blister rolled to break the seal and advance the solution to the detection station (probe deposits).
• the analyte and probe reagents were hybridized in the detection station at 42°C for 5min, while the SA-HRP conjugate in the second blister was preheated to 65°C.
• the conjugate blister was rolled, the seal broken, and the solution directed to the detection area to displace the analyte.
• the third blister containing the first wash solution preheated to 55°C was broken and the wash directed to the detection station and held there for 5min while the second wash solution was preheated to 55°C. Then the blister containing the second wash solution was broken and the wash directed to the detection station.
• the blister containing the dye signal-forming composition was rolled without preheating, and the seal broken, and the composition directed to the detection station where the color scores were read after a 5min incubation period using a color chart as described hereinbelow.
• the color scores are recorded in Table I and presented graphically in Figure 5A.
• the blister containing the analyte in each element was preheated to about 95°C for 120s and then rolled to break the seal and advance the solution to the area containing the four immobilized deposits of probe reagents, that is, the two control probes and the two HUT/HIV probes deposited with adhesive.
• probe reagents that is, the two control probes and the two HUT/HIV probes deposited with adhesive.
• the analyte and probe reagents were hybridized in the detection station at 42°C for 5min, while the blister containing the wash solution was preheated to 55°C. Then the wash solution blister was rolled to break the seal and direct the wash solution into the detection area to clean out the main channel and to remove unbound analyte from the detection area.
• the seal of the streptavidin/horseradish peroxidase conjugate blister was rolled and broken and the solution directed to the detection area where it binds to the immobilized biotinylated analyte over a 5min period.
• the second wash composition was preheated to 55°C, and the seal of the blister was then broken with the roller and directed to the detection station where it displaced the unbound label.
• the seal of the dye signal-forming composition in the last blister was broken with the roller, and the fluid directed to the detection station where it displaced the second wash solution.
• Dye formation on the probe deposits was allowed to proceed for 5 minutes before reading color density scores. The color of each probe deposit was evaluated by comparison of the wet dye density with a color chart where 0 is no density and 10 is the highest density. The color scores are recorded in Table II and presented graphically in the graph of Figure 5B.
• Example 2 it is shown below, in Example 2, that in fact no wash compartments are needed for correct and accurate functioning of the assay.
• the cuvettes in the second set were processed as follows:
• the analyte in the PCR blister was preheated to 95°C for 120s, and the blister was rolled to break the seal and direct the analyte to the 3 probe deposits in the detection station. Hybridization at 42°C was allowed to proceed for 5min while the SA-HRP solution in the second blister was preheated to 65°C. The second blister was then rolled to break the seal and the solution directed through the channels to the detection station. The conjugate was incubated over the detection station for 5min, then the blister containing the dye-forming detection dispersion was rolled without preheating to break the seal and direct the dispersion to the detection station to displace the SA-HRP.
• the 3-blister configuration allows for use of less reagents, a smaller unit manufacturing cost, less cuvette storage space, shorter processing times, and a smaller, less complex processor.

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• 1993
  • 1993-08-31 TW TW82107088A patent/TW313588B/zh not_active IP Right Cessation
  • 1993-10-21 DE DE1993617502 patent/DE69317502T2/de not_active Expired - Lifetime
  • 1993-10-21 EP EP19930202962 patent/EP0594260B1/en not_active Expired - Lifetime
  • 1993-10-21 DK DK93202962T patent/DK0594260T3/da active
  • 1993-10-22 FI FI934669A patent/FI934669A/fi unknown
  • 1993-10-22 JP JP26481393A patent/JP3795540B2/ja not_active Expired - Lifetime

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