WO2000029112A1 - One-step nucleic acid dipstick device with movable membrane - Google Patents

Abstract

The present invention relates generally to the field of nucleic acid detection and diagnostics. More particularly, the present invention relates to a disposable device for detection of specific nucleic acids in a sample. For example, the sample may be a DNA sample amplified using the polymerase chain reaction. The device is operated by aspirating the sample to be analyzed through a tube into a chamber. Inside the chamber, the sample is prepared by contact with pre-measured, pre-deposited reagents. The sample is then processed by mobilization via capillary action through a membrane having pre-measured, pre-deposited signal and/or detection reagents. The sample is analyzed by reading the results which develop on the membrane. For example, specific nucleic acids present in the sample may be detected colorimetrically via hybridization to complementary probes in the membrane. A major advantage of the device is that it provides one-step sample preparation, processing, and analysis. Further, the device is suitable for use outside of a controlled laboratory setting, such as at the point of care and/or in the field. Still further, the device requires no specialized skills to operate.

Description

ONE-STEP NUCLEIC ACID DIPSTICK DEVICE WITH MOVABLE MEMBRANE

This application claims the benefit of United States Patent Application No. 09/195,370 filed on November 18, 1998, the entire disclosure is herein incorporated by reference. The invention was made with government support under contract number 96-U 154400-000. Accordingly, the government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of nucleic acid detection and diagnostics. More particularly, the present invention relates to a disposable, one- step nucleic acid dipstick device for detection of specific nucleic acids in a sample-of- interest. In a preferred embodiment, the sample-of-interest is a polymerase chain reaction sample. Typically, the device is operated by aspirating the sample to be analyzed through a tube into a chamber. Inside the chamber, the sample is prepared by contact with pre-measured, pre-deposited reagents. The sample is then processed by mobilization via capillary action through a membrane having pre-measured, pre- deposited signaling and/or detection reagents thereon. The sample is analyzed by visually reading the results which develop on the membrane. A major advantage of the device is that it provides one-step sample preparation, processing, and analysis. Further, the device is suitable for use outside of a controlled laboratory setting, such as at the point of care or in the field.

BACKGROUND OF THE INVENTION

Dipstick devices have been widely used in the medical and veterinary diagnostic fields to test for the presence of various substances and activities in biological samples. Such devices have provided a rapid and convenient way to conduct various biological tests indicative of various physiologic and/or pathophysiologic conditions. Several examples are highlighted herein. A wide variety of dipsticks utilizing immunologic assays have been developed. For example, dipstick devices have been used in the following contexts: for the immunologic detection of oligoalbuminuria (see e.g. Kutter et al, 1995, Screening for oligoalbuminuria by means of Micral-Test^® II, a new immunological test strip, Eur. J. Clin. Chem. Clin. Biochem. 33, 243-245); for IGFBP-1 detection by immunochromatography (Rutanen et al., 1996, Evaluation of a rapid strip test for insulin-like growth factor binding protein- 1 in the diagnosis of ruptured fetal membranes, Clin. Chim. Acta 253, 91 -101 ); for the detection of hepatitis B virus by ELISA (Sumathy et al., 1992, A dipstick immunobinding enzyme-linked immunosorbent assay for serodiagnosis of hepatitis B and delta virus infections, J.

Virol. Meth. 38, 145-152); and for the detection of HIV by ELISA (Ray et al, 1997, An evaluation of dipstick-dot immunoassay in the detection of antibodies to HIV-1 and 2 in Zimbabwe, Trop. Med. Int'l Health 2, 83-88). Dipstick devices have also been used for immunoassay of urinary iϊbrin/fibrinogen associated with bladder cancer (Schmetter et al., 1997, A multicenter trial evaluation of the fibrin/fibrinogen degradation products test for detection and monitoring of bladder cancer, J. Urol. 158, 801 -805); for pancreatitis screening by immunochromatographic measurement of urinary trypsinogen-2 (Kemppainen et al., 1997, Rapid measurement of urinary trypsinogen-2 as a screening test for acute pancreatitis, Ne Engl. J. Med. 336, 1788- 1793); for monoclonal antibody-based enzyme immunoassay detection of PSA

(Madersbacher et al., 1996, Validation of a 10-minute dipstick test for serum prostate- specific antigen, Eur. Urol. 30, 446-450); and even for immunological detection of a fungal toxin (Saeger and van Peteghem, 1996, Dipstick enzyme immunoassay to detect Fusarium T-2 toxin in wheat, Appl. Environ. Microbiol. 62, 1880-1884).

Non-immunologic methods have also been developed for use in conjunction with dipstick devices. For example, a dipstick for the estimation of blood alcohol using a colorimetric saliva assay has been developed (see Schwartz et al, 1989, Evaluation of colorimetric dipstick test to detect alcohol in saliva: a pilot study, Ann. Emer. Med. 18, 1001/157-1003/159). Further, a dipstick device has been used for quantitive colloidal carbon particle detection of HCG (van Amerongen et al., 1994, Quantitative computer image analysis of a human chorionic gonadotropin colloidal carbon dipstick assay, Clin. Chim. Acta 229, 67-75); this assay combines a particle method with an immunologic method.

Further, a dipstick is available for many diagnostic and/or prognostic uses associated with clinical medicine. Examples include urinary LH detection, useful in timing ovulation (see Paz et al., 1990, Determination of urinary luteinizing hormone for prediction of ovulation, Gynecol. Obstct. Invest. 29, 207-210); and hepatitis C virus antibody detection and hepatitis B surface antigen detection (Mvere et al, 1996, Rapid and simple hepatitis assays: encouraging results from a blood donor population in Zimbabwe, Bull. World Health Org. 74, 19-24).

A dipstick hybridization method has been developed for detection of HIV- 1 (see Bawa et al., 1995, A non-radioisotopic reverse phase dipstick hybridization method for detection of polymerase chain reaction amplified product, Indian J. Med.

Res. 101, 142-146). However, this method requires multiple steps to perform, only works if digoxigenin-11-dUTP is incoφorated into the PCR-amplified product prior to detection, and is not appropriate for use outside of a controlled laboratory setting.

A one-step device incoφorating all necessary reagents for detection of a nucleic acid of interest and suitable for use in the field has not been previously described.

Rapidly-expanding nucleic acid sequence databases now exist which are highly relevant to understanding living organisms in normal and diseased states (see e.g., Heiter and Boguski, 1997, Science 278, 601-602; Holtzman et al, 1997, Science 278, 602-605). Nevertheless, such information is largely inaccessible to individuals lacking specialized training or experience. Accordingly, a need exists for analysis tools which can be easily used at the point of care or in the field to take advantage of this important knowledge resource. SUMMARY OF THE INVENTION The present invention relates generally to the field of nucleic acid detection and diagnostics. More particularly, the present invention relates to a disposable, one- step device for detection of specific nucleic acids in a sample. In a preferred embodiment, the sample comprises DNA amplified by polymerase chain reaction, ligase amplification reaction or rolling circle amplification. The device is operated by aspirating the sample to be analyzed through a tube into a chamber. Inside the chamber, the sample is prepared by contact with pre-measured, pre-deposited reagents. The sample is then processed by mobilization via capillary action through a membrane having pre-measured, pre-deposited signaling and/or detection reagents.

The sample is analyzed by reading the results which develop on the membrane.

The device combines all steps of a detection method into a user- friendly, integrated format compatible with use outside of a controlled laboratory setting, such as at the point of care or in the field. The device provides a versatile means for detection of specific nucleic acids, as described in detail below. A major advantage of the device is that it provides one-step sample preparation, processing, and analysis and requires no specialized skills to operate. Specific embodiments of the device are summarized below.

This invention provides a device for detection of a nucleic acid in a sample from a subject comprising: (a) a reaction chamber having reagents pre-deposited therein for preparation of the sample; and (b) a moveable membrane situated within the reaction chamber having reagents pre-deposited thereon for processing of the sample. In one embodiment, the reaction chamber comprises a flattened window area suitable for viewing the moveable membrane within. In another embodiment, the reaction chamber is attached to a fluid transfer tube. In yet another embodiment, the fluid transfer tube comprises a standpipe for preventing the sample from being ejected during positioning of the moveable membrane. The device may also comprise, either as part of the body of the device or as an attachment to the fluid transfer tube, an adaptor for attachment to a micro-well of a micro-PCR device. The fluid transfer tube may itself function as an aspiration tube or may be attached to a separate aspiration tube. In either case, the aspiration tube may be of a suitable diameter to fit within a capillary polymerase chain reaction tube.

In another embodiment, the aspiration tube has an outer diameter of from 100 micrometers to 2 millimeters. In a preferred embodiment, the aspiration tube has an outer diameter of from 200 micrometers to 1 millimeter. In yet another embodiment, the nucleic acid is amplified prior to detection by an amplification method. For example, the amplification method may be polymerase chain reaction, ligation amplification reaction, or rolling circle amplification. In another embodiment, detection of the nucleic acid in the sample is diagnostic for a disease or disorder, or the presence of an organism (e.g. bacterial, viral or fungal), in the subject. For example, the organism may include but is not limited to chlamydia, heφesvirus, Neisseria gonorrhea, human immunodeficiency virus (HIV), Epstein Barr virus (EBV), Helicobacter pylori, Haemophilus influenzae, Mycoplasma genitalium,

Clostridium perfringens, Clostridium botulinum, Bacillus anthracis, etc. Further, the disease or disorder may include but is not limited to tuberculosis, gonorrhea, acquired immunodeficiency syndrome (AIDS), botulism, anthrax, etc. Further, the nucleic acid detected may indicate the absence or presence of a disease or disorder having a genetic component, or the absence or presence of a propensity for such disease or disorder. For example, some forms of cardiovascular or neurologic disease may be associated with specific single nucleotide polymorphisms (SNPs). Such SNPs can be detected with the device of the invention. In another embodiment, the subject from which the sample is obtained is an animal or a plant. The subject may be a human subject. In another embodiment, the moveable membrane having reagents pre- deposited thereon comprises one or more immobilized probes. In another embodiment, the moveable membrane further comprises one or more probe markers for visibly marking the position of the one or more immobilized probes.

The present invention provides a device for detection of a nucleic acid in a sample from a subject comprising: (a) a reaction chamber having reagents pre- deposited therein for preparation of the sample; (b) a membrane situated within the reaction chamber having reagents pre-deposited thereon for processing of the sample; and (c) a membrane positioner for changing the position of the membrane within the reaction chamber. In one embodiment, the membrane comprises a material selected from the group consisting of nylon, nitrocellulose, paper, plastic and polyethersulfone.

In another embodiment, the membrane positioner is a plunger or a screw. In yet another embodiment, the reaction chamber comprises threads capable of threading the membrane positioner which positions the membrane by pushing on an absorbent material attached to the top of the membrane. In another embodiment, the reaction chamber is a barrel or tube. In yet another embodiment, the barrel is a 1 ml, 3 ml, 5 ml or 10 ml syringe barrel.

This invention provides a device for detection of a nucleic acid in a sample from a subject comprising: (a) a reaction chamber having reagents pre-deposited therein for preparation of the sample; (b) a fluid transfer tube attached to the floor of the reaction chamber for transferring the sample into the reaction chamber; (c) a membrane situated within the reaction chamber having reagents pre-deposited thereon for processing of the sample; and (d) a membrane positioner for dipping the membrane into the sample following sample preparation. In one embodiment, a micro-well PCR device adaptor is attached to the fluid transfer tube. In another embodiment, an aspiration tube is attached to the micro-well PCR device adaptor. In yet another embodiment, the membrane is constructed from a material selected from the group consisting of nitrocellulose, nylon, paper, plastic and polyethersulfone. In yet still another embodiment, the device has a plurality of probes immobilized on the membrane, wherein one or more of the plurality is capable of hybridizing under conditions of high stringency with the nucleic acid in the sample.

In another embodiment, the device has a plurality of probe markers selected from the group consisting of inks, dyes and stains visibly marking the position on the membrane of one or more of the plurality of probes. In another embodiment, a pre- deposited reagent carrier is situated on the membrane and comprises a polyfiberglass pad containing a signaling or detection reagent. In another embodiment, the signaling or detection reagent comprises 0.01 to 100 μm colored beads coated with avidin or streptavidin. In another embodiment, the nucleic acid is labeled with biotin. In another embodiment, a positive control site for color control is located on the membrane and comprises a biotinylated molecule, hi another embodiment, the reaction chamber is constructed from a material selected from the group consisting of polypropylene, polyethylene, polycarbonate, metal and glass. In another embodiment, the fluid transfer tube is constructed from a material selected from the group consisting of polypropylene, polyethylene, polycarbonate, polyimide, metal and glass. In another embodiment, a micro-well PCR device adaptor is attached to the fluid transfer tube and is constructed from a material selected from the group consisting of polypropylene, polyethylene, polycarbonate, metal and glass. In another embodiment, an aspiration tube is attached to the micro-well PCR device adaptor and is constructed from a material selected from the group consisting of polypropylene, polyethylene, polycarbonate, polyimide, metal and glass. In another embodiment, the reagents pre- deposited in the reaction chamber comprise one or more components suitable for adjustment of hybridization stringency. In another embodiment, the membrane positioner comprises a pressure regulator for aspiration of the sample into the reaction chamber. In another embodiment, the membrane positioner for dipping the membrane into the sample is selected from the group consisting of a plunger and a screw. In another embodiment, the reagents pre-deposited on the membrane comprise a microparticle selected from the group consisting of plastic, latex, carbon, magnetite and gold. In another embodiment, the microparticle ranges from 10 nm to 500 μm diameter. In another embodiment, the microparticle is labeled with a colored dye or a fluorescent label. In another embodiment, the microparticle is labeled with a protein selected from the group consisting of an enzyme, an antibody, a fluorescent protein, avidin and streptavidin. In another embodiment, the microparticle is labeled with a nucleic acid selected from the group consisting of deoxyribonucleic acid, ribonucleic acid and protein nucleic acid. In another embodiment, an absorbent material selected from the group consisting of cotton, felt, paper, polyfiberglass and wool is situated at the end of the membrane opposite the dipping end. In another embodiment, the membrane further comprises a pre-deposited reagent carrier having signaling or detection reagents pre-deposited thereon. In another embodiment, the pre-deposited reagent carrier is positioned on the membrane so as to be dipped into the sample following sample preparation. In another embodiment, one or more components suitable for adjustment of hybridization stringency is selected from the group consisting of inorganic salts, formamide and detergents. In another embodiment, the fluid transfer tube attached to the floor of the reaction chamber comprises a standpipe for preventing the sample from being ejected during positioning of the membrane. In another embodiment, the membrane comprises a plurality of probes immobilized thereon, a plurality of probe markers visibly marking the position of each of the plurality of probes, and a positive control site. In another embodiment, the pressure regulator is constructed from a material selected from the group consisting of rubber and silicone.

The present invention provides a device comprising: (a) a reaction chamber having pre-deposited reagents therein for preparation of a sample; and (b) a membrane positioned within the reaction chamber having pre-deposited reagents thereon for processing the sample. In one embodiment, the membrane is constructed from nylon, nitrocellulose or paper. The sample may be virtually any sample containing a nucleic acid. For example, the sample may be a cell or tissue sample (i.e. biopsy, buccal scrapings, hair and/or hair follicle, etc.) containing a nucleic acid. The sample may also be a nucleic acid sample amplified using the polymerase chain reaction (RT- PCR). In a preferred embodiment, detection of a nucleic acid in the sample is completed in less than two minutes. The device can be operated under a broad range of ambient temperatures and humidities at the point of care or in the field.

In one embodiment, the device is operated by aspirating the sample to be analyzed through a tube into a chamber. Inside the chamber, the sample is prepared by contact with pre-measured, pre-deposited reagents. The sample is then processed by mobilization via capillary action through a membrane having pre-measured, pre- deposited signaling and/or detection reagents. The sample is analyzed by reading the results which develop on the membrane. For example, specific nucleic acids present in the sample may be detected colorimetrically via hybridization to complementary probes immobilized on the membrane.

This invention provides a device for detection of a nucleic acid in a sample comprising: (a) a reaction chamber having reagents pre-deposited therein for preparation of the sample; (b) a membrane situated within the reaction chamber having signaling and/or detection reagents pre-deposited thereon for processing of the sample; and (c) positioning means for positioning the membrane within the reaction chamber.

By "on the membrane", it will be understood that signaling and/or detection reagents pre-deposited may actually be pre-deposited on a carrier (such as a "reagent pad") which is attached to the membrane (e.g. by an adhesive). Such a earner may comprise one or more materials similar to or different from the membrane material

(e.g. nylon, nitrocellulose, paper, plastic, polyethersulfone, polyfiberglass, felt, etc.). In one embodiment, the reaction chamber comprises a tube or barrel (e.g. a syringe barrel). In another embodiment, the membrane comprises a material selected from the group consisting of nylon, nitrocellulose, paper, plastic and polyethersulfone, etc. In another embodiment, the positioning means comprises a plunger. In yet another embodiment, the tube or barrel further comprises internal threads for threading a membrane positioner. In a preferred embodiment, the baπ'el is a 1 ml syringe barrel, a 3 ml syringe barrel, a 5 ml syringe barrel, a 10 ml syringe barrel, a 25 ml syringe barrel, or a 50 ml syringe barrel. In another embodiment, the membrane further comprises a positive control site, one or more immobilized oligonucleotide probes, one or more probe markers marking the position of said oligonucleotide probes, and one or more predeposited signaling and/or detection reagents. In yet another embodiment, the positioning means further comprises a membrane positioner having threads capable of threading with the reaction chamber. In another embodiment, the positioning means is a plunger. Further, this invention provides a device for detection of a nucleic acid in a sample comprising: (a) a membrane; (b) a plurality of oligonucleotide probes immobilized on the membrane; (c) a plurality of oligonucleotide probe markers visibly marking the position of each of the plurality of oligonucleotide probes on the membrane; (d) a signaling agent pre-deposited on the membrane at a position most distal to an absorbent material; (e) a positive control site for color control located most proximal to the absorbent material; (f) a reaction chamber; (g) a fluid transfer tube for transferring the sample into the reaction chamber; (h) a micro-well PCR device adaptor attached to the fluid transfer tube; (i) an aspiration tube attached to the micro-well PCR device adaptor; (j) one or more sample preparation reagents pre- deposited on the floor of the reaction chamber; (k) a pressure regulator for application of negative pressure in the reaction chamber; and (1) a membrane positioner for moving the membrane relative to the reaction chamber. In one embodiment, the membrane is constructed from a material selected from the group consisting of nitrocellulose, nylon, paper, plastic and polyethersulfone. In another embodiment, the plurality of oligonucleotide probes immobilized on the membrane is capable of hybridizing under conditions of high stringency with a nucleic acid of interest. In yet another embodiment, the plurality of oligonucleotide probe markers visibly marking the position on the membrane of each of the plurality of oligonucleotide probes is selected from the group consisting of inks, dyes and stains. In yet still another embodiment, the signaling agent pre-deposited on the membrane at a position most distal to an absorbent material comprises 0.01 to 100 μm colored beads or microparticles coated with avidin, streptavidin or NeutrAvidin (Product #31000, Pierce Chemical Company, Rockford, Illinois). In a preferred embodiment, the nucleic acid sample is labeled with biotin. In another embodiment, the positive control site for color control located most proximal to the absorbent material comprises a biotinylated molecule immobilized on the membrane. In another embodiment, the reaction chamber is constructed from a material selected from the group consisting of polypropylene, polyethylene, polycarbonate, metal and glass. In another embodiment, the fluid transfer tube for transferring the sample into the reaction chamber is constructed from a material selected from the group consisting of polypropylene, polyethylene, polyimide, polycarbonate, metal and glass. In yet another embodiment, the micro-well PCR device adaptor attached to the fluid transfer tube is constructed from a material selected from the group consisting of polypropylene, polyethylene, polycarbonate, metal and glass. In yet still another embodiment, the aspiration tube attached to the micro-well PCR device adaptor is constructed from a material selected from the group consisting of polypropylene, polyethylene, polyimide, polycarbonate, metal and glass. In a preferred embodiment, the sample preparation reagents pre-deposited on the floor of the reaction chamber comprise inorganic salts and/or other components (e.g. formamide, detergents, etc.) suitable for adjustment of hybridization stringency. In another embodiment, the pressure regulator for application of negative pressure in the reaction chamber is constructed from a material selected from the group consisting of rubber and silicone. In another embodiment the membrane positioner for moving the membrane relative to the reaction chamber is selected from the group consisting of a plunger and a screw. In another embodiment, the signaling agent pre-deposited on the membrane at a position most distal to an absorbent material comprises a microparticle selected from the group consisting of plastic, latex, carbon, magnetite and gold. In another embodiment, the microparticles range from 100 nm to 500 μm in diameter. In yet another embodiment, the microparticle is labeled with a visual dye or a fluorescent label. In another embodiment, the microparticle is labeled with a protein selected from the group consisting of an enzyme, an antibody, a fluorescent protein, avidin and streptavidin. In yet another embodiment, the microparticle is labeled with a nucleic acid selected from the group consisting of deoxyribonucleic acid, ribonucleic acid and protein nucleic acid. In another embodiment, the absorbent material is selected from the group consisting of cotton, felt, paper, polyfiberglass and wool.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a nucleic acid dipstick device of the invention; FIG. IB depicts a section through a-a in FIG. 1A. FIG 2. illustrates a device of the invention attached to a PCR device for sample loading.

FIG. 3 illustrates a well known "lateral flow" method of nucleic acid detection which may be used in the device of the invention.

FIG. 4 illustrates a device of the invention having a bulb-type pressure regulator for aspirating the sample and a flattened window area for viewing the membrane; FIG. 4B depicts a section through A-A in FIG. 4A viewed from the top of the device looking down.

DETAILED DESCRIPTION OF THE INVENTION

The device of the invention described herein is suitable for detection of specific nucleic acid present in a test sample. The device typically comprises a disposable reaction chamber with a membrane situated inside. The reaction chamber contains pre-deposited reagents for sample preparation. The membrane contains pre- deposited reagents for sample processing. Such pre-deposited reagents "on the membrane" may actually be pre-deposited on a carrier (e.g. "reagent pad") which is attached to the membrane (e.g. by an adhesive). Such a carrier may comprise one or more materials similar to or different from the material used to manufacture the membrane itself (e.g. nylon, nitrocellulose, paper, plastic, polyethersulfone, felt, etc.). The device typically provides a means for placement of a test sample to be analyzed within the chamber, such as by aspiration of the sample. The membrane is situated within the chamber so as to not interfere with the pre-deposited reagents for sample preparation. Further, the membrane has pre-deposited reagents for sample processing, including specific detection means for detection of one or more nucleic acids of interest. Such specific detection means of the membrane may be any nucleic acid detection means known to those skilled in the art of nucleic acid detection. A device of the invention may be used as follows. Briefly, a test sample is aspirated into a chamber where it is prepared by contacting pre-measured, pre- deposited reagents. The test sample so prepared is then processed by contacting a membrane situated within the chamber having specific nucleic acid detection means pre-deposited thereon. Analysis of the processed sample for detection of one or more specific nucleic acids is achieved by inspection of the membrane. In a preferred embodiment, such inspection is visual inspection under standard indoor or outdoor visible light.

Dipstick Device

A one-step device of the invention 10 typically comprises a reaction chamber and a membrane situated inside, wherein the reaction chamber contains pre-deposited reagents for sample preparation and the membrane contains pre-deposited reagents for sample processing, including signaling and/or detection reagents and/or membrane- bound probes for recognizing specific nucleic acids of interest. However, a one-step device of the invention 10 can have many additional features. A preferred embodiment of the invention is illustrated in FIG. 1 A; section a-a through the device illustrated in FIG. 1A is shown in FIG. IB. Components of such a device 10 may include but are not limited to the following: a membrane positioner 12 for moving the membrane; a pressure regulator (i.e. plunger) 14 for aspiration of a sample; an absorbent material 16 for capturing a sample after it has traveled along the membrane; a positive control site 18 for insuring optimum performance of the device (e.g. color control); one or more membrane-bound probes 20 for detection of specific nucleic acids of interest; one or more probe markers 22 for visibly marking the position on the membrane of the membrane-bound probes; a moveable membrane 24 for sample processing; one or more pre-deposited reagents residing in a reagent carrier or pad 26 attached to the membrane; a reaction chamber 28 containing the moveable membrane; reagents for sample preparation 30 pre-deposited on the floor of the reaction chamber; a fluid transfer tube 32 for loading the reaction chamber, which tube may protrude above the floor of the reaction chamber so as to form a standpipe which prevents escape of an aspirated sample; a micro-well PCR adaptor 34 for attachment of the device directly to a micro-well PCR thermal cycler; and an aspiration tube 36 of a suitably narrow outer diameter for accessing a sample to be loaded from a narrow container.

With reference to the embodiment of the device as illustrated in FIG. 1 , a fluid sample to be analyzed is placed in the reaction chamber 28 using the aspiration tube 36 and the membrane positioner 12 to displace the pressure regulator 14 upward, so as to create a negative pressure within the reaction chamber to aspirate the sample. The reaction chamber may be, e.g., a barrel, a tube, a flattened cylinder, etc. A suitable container provides a transparent area for viewing the membrane within. The upper opening of the fluid transfer tube 32 may be elevated above the floor of the reaction chamber 28 to form a standpipe which creates a space within the chamber for sample preparation (i.e mixing with pre-deposited reagents). In this embodiment, the fluid sample aspirated into the reaction chamber 28 is prevented from subsequently exiting the reaction chamber 28 when the membrane is positioned using a membrane positioner 12 attached to a pressure regulator 14. The aspiration tube 36 may be, e.g., a needle, a catheter, polyethylene tubing, and/or polyimide tubing. In a preferred embodiment, the reaction chamber 28 and fluid transfer tube 32 are made of a transparent polymer material (e.g. polycarbonate, polyethylene, polypropylene, polystyrene, etc.). Further, a micro-well adaptor 34 may be used to directly attach the device to a sample well of a micro-PCR device.

Once a sample is delivered into the reaction chamber, the sample is allowed to mix with the pre-deposited reagents 30 to effectuate sample preparation. Such sample preparation may, inter alia, aid in hybridization and/or signal detection. The moveable membrane 24 situated within the reaction chamber comprises reagents thereon, including specific nucleic acid probes, signaling and/or detection reagents. Such reagents may include one or more immobilized oligonucleotides capable of hybridizing with a specific nucleic acid of interest under various stringency conditions. In the embodiment illustrated in FIG. 1, the moveable membrane 24 is dipped into an aspirated sample which has already mixed with the reagents pre- deposited on the floor of the reaction chamber. The membrane may be moved using a membrane positioner 12 functioning as a screw as illustrated in FIG. 1., or as a plunger. The membrane may also be moved manually (see FIG. 4). Processing of a prepared sample occurs during capillary flow of the sample along the membrane 24 from the dipped end to the absorbent material or wick 16 suitable for trapping liquid.

During flow along the membrane, the sample comes into contact with one or more pre-deposited reagents including signaling and/or detection reagents situated on a reagent earner or pad 26 and one or more pre-deposited specific probes 20 immobilized on the membrane. Here, a signaling and/or detection reagent may comprise any reagent capable of generating a detectable signal. Such reagents may include but are not limited to the following: visually detectable dyes or fluorescently- labeled microparticles (e.g., latex, carbon, magnetic, or other microparticles); colloidal gold or carbon; protein complexes (e.g., enzymes, antibodies, fluorophores, etc.); and DNA, protein nucleic acid (PNA), or RNA reagents used singly or in combination. By contrast, pre-deposited reagents functioning as specific probes on the membrane may include but are not limited to immobilized DNA, PNA, or RNA oligonucleotide probes (or any combination and/or derivative thereof) which can hybridize to a specific target nucleic acid of interest.

A nucleic acid in the sample may be detected by specifically hybridizing to an immobilized probe 20 on the membrane. Specific hybridization events may be visualized using any method known to one skilled in the art. For example, a colored signal may be detected next to a probe marker 22 which marks the location of a specific immobilized probe on the membrane. Such a probe marker can be any of the following: colored dyes, inks, paints, or other visually detectable substance which, when placed on the membrane, will allow the observer to distinguish one specific probe site from another, both before and after signal development takes place, but does not interfere with signal development or the determination of signal. Pre- deposited specific probes will typically include positive controls 18 for hybridization and/or detection events, which may be located at any position on the membrane. In a preferred embodiment, such positive controls are located distal to the dipped end of the membrane to insure that reagent depletion has not occurred prior to contact with the specific probes (see e.g. FIG. 1).

A device of the invention 10 attached to a PCR device 38 is illustrated in FIG. 2. As shown in FIG. 2, the device may be used to directly sample a capillary PCR reaction 42 containing amplified nucleic acids without removing the capillary from the thermal cycling chamber 40. Also shown in FIG. 2 is a pressure regulator 14, absorbent material (i.e. a wick) 16, and a reagent carrier or pad 26. In the reagent carrier 26 shown in FIG. 2, the black circles represent colored beads which are mobilized along the membrane by fluid capillary action as illustrated in FIG. 3 for detection of specific nucleic acid sequences. The detection method illustrated in FIG. 3 is suitable for use with a device of the invention. Such detection methods are described in further detail below.

Another embodiment of a device of the invention is illustrated in FIG. 4. In this embodiment, the pressure regulator for aspiration of a sample takes the form of a rubber bulb 46 which is not attached to a membrane positioner. Here, positioning of the membrane is accomplished manually, as described in further detail below below.

Generally, the reagents which may be used in the device of the invention can include any reagent known to be useful in the detection of nucleic acids. Many such reagents exist and are well described in the art. Such reagents include but are not limited to hybridization salts, oligonucleotides and analogs thereof, denaturants of double-stranded target nucleic acids (e.g. chemical denaturants), and methods for visualization of a hybridization signal (e.g. colored beads or other particles, or enzymes such as luciferase, alkaline phosphatase, etc.). Further examples of suitable reagents are provided herein in the Sections which follow. In a preferred embodiment, streptavidin-coated colored beads are attached directly to biotin-labeled, amplified nucleic acids in a sample to be analyzed. An adapter may be used for attachment of the device of the invention to a micro-PCR well prior to sample aspiration. In a preferred embodiment, such an adapter may have a conical shape and dimensions of 0.200 inches long, or longer, by 0.100 inches in diameter, tapering to 0.080 inches in diameter.

Nucleic acid detection may be rapidly accomplished using the device of the invention. In one embodiment, detection of a nucleic acid of interest is completed in from about 1 second to about 60 minutes. In another embodiment, detection of a nucleic acid of interest is completed in from about 10 seconds to about 10 minutes. In yet another embodiment, detection of a nucleic acid of interest is completed in two minutes or less.

The device may be used over a broad range of ambient temperatures and humidities. Generally, suitable ranges of temperature and humidity will be determined by the detection means employed within the device. The device itself may be constructed to physically withstand from 0^UC to 100°C and from 0% to 100% humidity. Nucleic acid detection means may be designed to operate over a broad range of temperatures and humidities by adjusting the stringency of hybridization using techniques well known in the art. In one embodiment, the operating temperature and humidity for the device and the nucleic acid detection means employed therein may range from 0°C to 80°C and from 0% to 100%, respectively. In another embodiment, the operating temperature and humidity for the device and the nucleic acid detection means employed therein may range from 5°C to 70°C and from 5% to 95%, respectively. In yet another embodiment, the operating temperature and humidity for the device and the nucleic acid detection means employed therein may range from 10°C to 55°C and from 10% to 90%, respectively. In a preferred embodiment, the operating temperature and humidity for the device and the nucleic acid detection means employed therein may range from 15°C to 40°C and from 15% to 85%, respectively. Membrane

A membrane of the invention, which comprises means for detection of a nucleic acid of interest, is situated within the reaction chamber in a pre-made, ready- for-use format. The membrane may be constructed from a wide variety of materials well known in the art. The material chosen may be any material which is suitable for pre-deposit of probes or reagents, capable of permitting capillary action, and capable of being positioned either manually or by a membrane positioner. For example, nitrocellulose, nylon, paper, porous plastic, polyethersulfone, etc., may be used. A moveable membrane 24 may comprise any number of discrete reagents, including but not limited to: one or more positive control sites 18, one or more immobilized nucleic acid probes 20 and probe markers 22, and one or more reagent carriers or pads 26 (see FIGS. 1-3).

Further, the "membrane" may take on a variety of forms and need not be embodied as a rectangular flat sheet. For example, a solid cylinder having pre- deposited reagents thereon may be used. Further, a hollow cylinder having pre- deposited reagents therein is provided, such as a standard capillary tube open at both ends. When using such a hollow cylinder, reagents pre-deposited therein may be suspended in a gel within the hollow cylinder or deposited on the inner or outer wall of the hollow cylinder.

Pre-Deposited Reagent Carrier

In a preferred embodiment, the membrane of the invention comprises, as an integral component thereof, one or more pre-deposited reagent carriers (e.g. "reagent pads") attached thereto. Such carriers provide flexibility in manufacturing a device of the invention suitable for detection of various nucleic acids. A reagent carrier may be constructed from a wide variety of materials well known in the art. The material chosen may be any material which is suitable for pre-deposit of reagents and capable of being affixed to the membrane. For example, polyfiberglass, cellulose, blotting paper, porous plastic material, polyethersulfone, etc., may be used. In a preferred embodiment, a polyfiberglass or blotting paper reagent carrier is used. A reagent carrier of the invention may be attached to the membrane at any position. In a preferred embodiment, a reagent carrier carrying one or more reagents to be mobilized along the membrane is attached at the end of the membrane to be dipped into the sample following sample preparation. Attachment of a reagent carrier to a membrane may be earned out using any method known in the art. Such methods include but are not limited to adhesives, glues, clamps, clips, staples, VELCRO'⁹, and the like.

A reagent carrier may be used to provide any reagent suitable for use in the device of the invention. For example, hybridization salts, oligonucleotides or analogs or derivatives thereof, denaturants, colored particles, enzymes, etc., may be provided on the membrane via a reagent carrier.

Reaction Chamber A reaction chamber of the invention contains pre-deposited reagents for sample preparation. Further, a reaction chamber can accommodate a moveable membrane having pre-deposited reagents thereon for sample processing. The reaction chamber of the invention may additionally comprise a fluid transfer tube for loading a sample and, in a preferred embodiment, a standpipe for preventing sample unloading when reaction chamber pressure is increased (see FIG. 1 ). In one embodiment, the reaction chamber is a tube or a barrel. In a preferred embodiment, a portion of the reaction chamber 28 may be slotted 44 to create a flat window for viewing the membrane (see FIG. 4). A section through A-A in FIG. 4A is illustrated in FIG. 4B looking down the length of a reaction chamber toward the aspiration tube of the device. A reaction chamber of the invention may be designed from inexpensive materials, e.g., a disposable Pasteur pipet, pipet tip or ball point pen body. Virtually any elongated hollow container may be fashioned to contain the device of the invention.

Pressure regulation in a reaction chamber of the invention for sample loading via aspiration may be achieved using a plunger, a screw, or a bulb. In a preferred embodiment, a screw-type device 12 having a rubber or silicone gasket 14 is used to regulate reaction chamber pressure (see e.g. FIG. 1 ). In another preferred embodiment, a bulb 46 made of flexible material (e.g. rubber) is used to control reaction chamber 28 pressure (see FIG. 4). When using a bulb-type pressure regulation with a reaction chamber not having a standpipe, as shown in FIG. 4, the sample is first aspirated into the chamber using the bulb. The membrane is then positioned into the sample by capping the aspiration tube 36 in an airtight fashion, removing the bulb 46 to allow access to the top of the moveable membrane, and pushing the membrane down into the sample using a suitable implement (e.g. a finger, a pencil, a paper clip, etc.). Using a suitably flexible bulb, the membrane may even be pushed down into the sample without removing the bulb, so long as the aspiration tube remains capped in an airtight fashion so as to preclude ejecting the sample. The latter method of using a device of the invention is preferred since it insures that no contaminants are allowed to enter the reaction chamber prior to signal development.

Absorbent Material

An absorbent material or wick 16 (see FIGS. 1-3) is used for trapping a sample that has been prepared and processed in a device of the invention. Such trapping occurs after the sample has traveled along the membrane under capillary action. The absorbent material may be any material capable of absorption. Such material may include, but is not limited to, cotton, felt, paper, polyfiberglass and wool. The absorbent material may be attached to the membrane by any method known in the art, including but not limited to, adhesive or glues, staples, VELCRO^®, and the like.

Detection Methods

A wide variety of detection means may be used in conjunction with the device of the invention. Well known nucleic acid detection methods suitable for use with the device include, but are not limited to, lateral flow methods using bead-based detection systems, hybridization, antibody-based nucleic acid detection, and the like. Further, methods of detection may include but are not limited to visual (e.g. by colored beads, a precipitating substrate, etc.), fluorescent (e.g. fluorescein, rhodamine, green fluorescent protein, etc.), chemiluminescent (e.g. luciferin luciferase assay) and electrochemical (i.e. a change in potential or charge).

Capillary action flow methods using bead-based detection systems are well known in the art. Such methods are preferred for use as a nucleic acid detection means in the device of the invention (see e.g. Zaun et al., 1995, United States Patent No. 5,415,839, Apparatus and method for amplifying and detecting target nucleic acids, issued May 16, 1995, which is incorporated by reference herein in its entirety).

One such method is schematically illustrated in FIG. 3. Here, an amplified nucleic acid sample ("PCR product" in FIG. 3) is denatured prior to contact with a support (i.e. membrane) having immobilized nucleic acid probes attached thereto. Denaturation may be accomplished by any method known in the art. The support illustrated in FIG. 3 is suitable for use as a membrane in the device of the invention. In the example depicted, the membrane is capable of detecting two specific target sequences (i.e. "Target 1 " and "Target 2" in FIG. 3). Further, colored beads provide a suitable means of visualizing a positive signal, as illustrated in this example (i.e. "Beads" in FIG. 3). Finally, the location of an absoφtive material to capture sample fluid which has flowed along the membrane is shown (see "Wick" in FIG. 3) and a preferred location on the membrane for a positive color-development control is also shown (see "Positive Control" in FIG. 3). In the example shown (lower right portion of FIG. 3), a positive signal has developed at the "Target 2" position but not at the "Target 1 " position, and the positive control indicates that the detector has performed adequately. Thus, in the FIG. 3 example shown, it may be concluded that the PCR product contains the target 2 sequence but not the target 1 sequence.

Another well known method which may be used as a nucleic acid detection means in the device of the invention is termed Genetic Bit Analysis (GBA). Briefly,

GBA is a method for typing single nucleotide polymoφhisms in DNA (see Nikiforov et al., 1994, Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms, Nucl. Acids Res. 22, 4167-4175, which is incoφorated herein by reference in its entirety; see also Nikiforov et al, U.S. Patent No. 5,679,524, which is incoφorated herein by reference in its entirety). In this method, a "genetic bit" refers to the most elementary unit of genetic information, namely, a single nucleotide. The

GBA method is highly flexible and can be applied under a wide variety of biochemical conditions to the typing of any nucleic acid polymorphism for which the nucleotide sequence is known (see Figure 1 of Nikiforov et al., Nucl, Acids Res., Id., for a schematic representation of single nucleotide typing by GBA). For use in the device of the invention, the GBA method is performed using one or more GBA nucleic acid probes (a.k.a. GBA primers or immobilized oligonucleotide primers) immobilized on the membrane of the device (e.g., see 20 of FIG. 1 herein) instead of on a 96 well polystyrene plate (see Nikiforov et al., Id.).

A suitable GBA hybridization buffer may comprise a final concentration of 1.5

M NaCl and 10 mM EDTA adjusted to pH 7.4-7.6, where EDTA is ethylenediaminetetraacetic acid. When using a lateral flow format for nucleic acid detection, hybridization time can be as brief as the time required for lateral flow itself. Hybridization may also be carried out, e.g., from 1 minute to 30 minutes at room temperature. The GBA hybridization buffer may also be modified to include 1 mM

CTAB and 0.167%o Tween-20 (vol. /vol.), where CTAB is cetyltrimethylammonium bromide.

Immobilization of specific GBA primers on the membrane of a device of the invention may be performed by any technique known in the art (e.g. baking, chemical crosslinking, etc.). For example, where the membrane is made of nitrocellulose, the GBA primers may be immobilized thereon by baking at 80°C for 2 hours. Further methods for immobilization of nucleic acids may be found in standard reference texts (see e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; see also,

Ausubel et al., eds., in the Current Protocols in Molecular Biology series of laboratory technique manuals, 1987-1997 Current Protocols, 1994-1997 John Wiley and Sons, Inc.). During manufacture, a TNTw solution can be used to wash the membrane of the device following immobilization of specific GBA primers thereon; this solution contains 10 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.05% Tween-20 (vol./vol.).

A variety of other well known methods may be used as nucleic acid detection means in the device of the invention. Such methods include but are not limited to the following examples. An amplified hybridization assay may be used (see Schneider et al., 1989, U.S. Patent No. 4,882,269). Here, a family of signal-generating secondary probes bind to a primary probe that hybridizes to the target sequence of interest, thereby generating an enormously amplified signal. Further, a sandwich hybridization assay may be used (see Ranki et al., 1986, U.S. Patent No. 4,563,419; Dunn et al, 1977, Cell 12, 23-36). The sandwich hybridization technique utilizes two complementary nucleic acid reagents specific for each target nucleic acid to be identified. Any probe known to one skilled in the art may be used in the device of the invention (see e.g. Dunn et al., 1980, Meth. Enzymol. 65, 468-478; Orum et al., 1993, Nucl. Acids Res. 21, 5332-5336; Carlsson et al., 1996, Nature 380, 207-207; and Castro and Williams, 1997, Anal. Chem. 69, 3915-3920).

Troubleshooting

The device of the invention described herein does not require maintenance or troubleshooting. On the contrary, the device is designed to be self-contained nucleic acid detection device suitable for use at the point of care or "in the field" by individuals not having any specialized training or expertise in nucleic acid detection.

Further methods of use with the device

Any method or reagent known to one skilled in the art may be used together with the device of the invention. For example, numerous nucleic acid detection methods and reagents are well known in the art and can be used in, or together with, the disclosed device. Several examples of such methods and reagents follow in the sections below.

_J Adjusting Stringency

Detection methods suitable for use in connection with the device of the invention include nucleic acid hybridization under low, moderate, or high stringency conditions. Methods for adjustment of hybridization stringency are well known in the art (see e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed.,

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; see also, Ausubel et al., eds., in the Current Protocols in molecular Biology series of laboratory technique manuals, 1987-1994 Current Protocols, 1994-1997 John Wiley and Sons, Inc.; see especially, Dyson, N.J., 1991 , Immobilization of nucleic acids and hybridization analysis In: Essential Molecular Biology: A Practical Approach, Vol. 2,

T.A. Brown, ed., pp. 1 1 1-156, IRL Press at Oxford University Press, Oxford, U.K.; each of which is incorporated by reference herein in its entirety). Salt concentration, melting temperature, the absence or presence of denaturants, and the type and length of nucleic acid to be hybridized (e.g. DNA, RNA, PNA) are some of the variables considered when adjusting the stringency of a particular hybridization reaction according to methods known in the art.

Nucleic Acid Amplification

The polymerase chain reaction (PCR) may be used in connection with the device of the invention to amplify a desired sequence from a source (e.g., a tissue sample, a genomic or cDNA library) prior to detection. Oligonucleotide primers representing known sequences can be used as primers in PCR. PCR may be earned out by use of a thermal cycler (e.g., from Perkin-Elmer Cetus) and a thermostable polymerase (e.g., Gene Amp™ brand of Taq polymerase). The nucleic acid being amplified may include but is not limited to mRNA, cDNA or genomic DNA from any species. The PCR amplification method is well known in the art (see e.g., U.S. Patent Nos. 4,683,202; 4,683,195 and 4,889,818; Gyllenstein et al., 1988, Proc. Nat'l Acad. Sci. U.S.A. 85, 7652-7656; Oclrman et al., 1988, Genetics 120, 621-623; Loh et al., 1989, Science 243, 217-220). The ligase amplification reaction (LAR) method may also be used for nucleic acid amplification in connection with the invention (see Wu et al., 1989, Genomics 4, 560-569; Nikiforov et al., U.S. Patent No. 5,679,524, issued October 21, 1997).

The rolling circle amplification (RCA) method may also be used for nucleic acid amplification in connection with the invention. One such method utilizing rolling circle replication by DNA polymerase under isothermal conditions has recently been described by Lizardi et al. (1998, Nature Genetics 19, 225-232; see also references therein).

Any prokaryotic cell, eukaryotic cell, or virus, can serve as the nucleic acid source. For example, nucleic acid sequences may be obtained from the following sources: human, porcine, bovine, feline, avian, equine, canine, insect (e.g., Drosophila), invertebrate (e.g., C. elegans), plant, etc. The DNA may be obtained by standard procedures known in the art (see e.g., Sambrook et al., 1989, Molecular

Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II).

Oligonucleotide Analogs

Nucleic acids used in conjunction with the device of the invention are often oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects, an oligonucleotide is 10 nucleotides, 15 nucleotides, 20 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 500 nucleotides, or 1000 nucleotides in length. An oligonucleotide can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, or single-stranded or double-stranded, or partially double-stranded. An oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, or a combination thereof. An oligonucleotide may include other appending groups, such as biotin, fluorophores, or peptides. An oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2thiouridine, 5- carboxyiriethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,

N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2- methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5- methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.

An oligonucleotide may comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

An oligonucleotide may comprise at least one modified phosphate backbone selected from the group including but not limited to a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phsophoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

An oligonucleotide or derivative thereof used in conjunction with the device of the invention may be synthesized using any method known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16, 3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Nat'l Acad. Sci. U.S.A. 85, 7448-7451), etc. An oligonucleotide may be an α-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (see Gautier et al., 1987, Nucl. Acids Res. 15, 6625-6641).

Oligonucleotides may be synthesized using any method known in the art (e.g., standard phosphoramidite chemistry on an Applied Biosystems 392/394 DNA synthesizer). Further, reagents for synthesis may be obtained from any one of many commercial suppliers. An exemplary supplier with an outstanding selection of suitable reagents is Glen Research in Sterling, Virginia. For example, an abasic linker may be introduced into a modified oligonucleotide at an uncertain position using Spacer Phosphoramidite C-, from Glen Research.

Oligonucleotides used in connection with the device of the invention will often be so modified, e.g. for detection, labeling, hybridization, etc. Further exemplary modifications and their uses are set forth below.

2'-Ome-RNA phosphoramidites may be used in the manufacture of suitable oligonucleotides for use with the device of the invention and may be obtained from commercial suppliers (e.g. Glen Research, Sterling, Virginia). 2'-Ome-RNA CE (β- cyanoethyl) phosphoramidites are designed to produce synthetic oligonucleotides containing nuclease-resistant 2'-O-methyl ribonucleotide linkages. Deprotection, isolation and handling of 2'-O-methyl oligonucleotides are identical to procedures used for ohgodeoxynucleotides. Such phosphoramidites include but are not limited to the following: 2'-Ome-A-CE phosphoramidite, 2'-Ome-C-CE phosphoramidite, 2'-

Ome-G-CE phosphoramidite and 2'-Ome-U-CE phosphoramidite, where A, C, G, and U designate adenine, guanine, cytosine and uracil, respectively.

Such modified bases, when incoφorated into an oligonucleotide, also prevent self-hybridization-based extension since these bases cannot be extended. For example, a capture oligonucleotide made up of DNA and Ome-RNA bases requires at least 4 to 7 DNA bases at the 3' end to act as an extension initiator. These modified bases do not affect hybridization itself to any significant degree.

3'-Phosphate CPG is 2-[2-(4,4'-dimethoxytrityloxy)- ethylsulfonyl] ethyl- succinoyl-long chain alkylamino-CPG, also available from Glen Research. It may be used as an alternative to enzymatic techniques to carry out phosphorylation of an oligonucleotide at the 3'-terminus during oligonucleotide synthesis.

Use of a 3'-phosphorylated oligonucleotide as a competitive binding oligonucleotide (CBO) is preferred since 3'-phosphorylated oligonucleotides cannot be extended when included in a PCR reaction mixture. In this way, only unphosphorylated primer included in a reaction mixture are extended during DNA amplification (i.e. only the primers which are intended to be extended). Other oligonucleotide modifications, such as dideoxy base CPG, may be used to perform the same function (also available from Glen Research).

Spacer phosphoramidite molecules may be used during oligonucleotide synthesis, e.g., to bridge sections of oligonucleotides where base pairing is undesired or to position labels or tags away from an oligonucleotide portion undergoing base pairing. The spacer length can be varied by consecutive additions of spacer phosphoramidites. Spacers phosphoramidite molecules may be used as 5'- or 3'- oligonucleotide modifiers. Such spacers include Spacer Phosphoramidite 9 (i.e. 9-0- Dimethoxytrityl- triethyleneglycol, l -[(2-cyanoethyl)-(N, N-diisopropyl)]- phosphoramidite, and Spacer Phosphoramidite 18 (i.e. 18-0-Dimethoxytrityl- hexaethyleneglycol, l-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), both available from Glen Research (Sterling, Virginia).

Other spacers are available for use in standard oligonucleotide synthesis. For example, Spacer Phosphoramidite C3 and dSpacer Phosphoramidite can be used to destabilize undesirable self-hybridization events within capture oligonucleotides or to destabilize false hybridization events between incorrectly-matched template/probe complexes. Such spacers, when positioned at the 3' end of an oligonucleotide, will also prevent incorrect extension products from being generated when included in a PCR reaction mixture.

One spacer available from Glen Research , Spacer Phosphoramidite C3 (i.e. 3-

O-Dimethoxytrityl-propyl-1 -[(2-cyanoethyl)-(N, N-diisopropyl)] -phosphoramidite), can be added to substitute for an unknown base within an oligonucleotide sequence.

A branching spacer may be used as one method to increase label incoφoration into an oligonucleotide. Such a branching spacer may also be used to increase a detectable signal by hybridization through multiply branched capture probes or PCR primers. Branching spacers are available commercially, e.g., from Glen Research.

Biotinylated oligonucleotides are well known in the art. An oligonucleotide may be biotinylated using a biotin-NHS ester procedure. Alternatively, biotin may be attached during oligonucleotide synthesis using a biotin phosphoramidite (Cocuzza, 1989, Tetrahed. Lett. 30, 6287-6290). One such biotin phosphoramidite available from Glen Research is l-Dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-0- (2-cyanoethyl)-(N, N-diisopropyl)-phosphoramidite. This compound also has a branch point to allow further additions. The branched spacer used in this biotin phosphoramidite has been described by Nelson et al. (1992, Nucl. Acids Res. 20, 6253-6259).

Another 5'-biotin phosphoramidite, namely [l -N-(4, 4'-Dimethoxytrityl)- biotinyl-6-aminohexyl]-2-cyanoethyl-(N, N-diisopropyl)-phosphoramidite, may be used to biotinylate an oligonucleotide. This compound is sold by Glen Research under license from Zeneca PLC.

Fluorescent dyes may also be incoφorated into an oligonucleotide using dye- labeled phosphoramidites. Two such labels are 5'-Hexachloro-Fluorescein Phosphoramidite (HEX), and 5'-Tetrachloro-Fluorescein Phosphoramidite (TET), both available from Glen Research.

Peptide Nucleic Acid Peptide nucleic acid (PNA) may be used in connection with the device of the invention. PNA was first described in 1991 by Nielsen et al. (1991, Science 254, 1497-1500; see also Egholm et al., 1992, J. Am. Chem. Soc. 1 14, 1895-1897; Egholm et al., 1993, Nature 365, 566-568). Briefly, PNA is a nucleic acid mimetic having a neutral peptide-like backbone instead of a negatively-charged sugar-phosphate backbone. However, the same nitrogenous bases (i.e. adenine, guanine, cytosine and thymine) are used in PNA as found in DNA and RNA. Consequently, PNA undergoes Watson-Crick base pairing with DNA and RNA. The amino terminal end of PNA is equivalent to the 5' end of DNA. Generally, PNA is not recognized as a substrate for DNA polymerases, nucleic acid binding proteins, or other enzymes, including proteases and nucleases. However, some analogs may be recognized (see e.g. Lutz et al, 1997, Recognition of uncharged polyamide linked nucleic acid analogs by DNA polymerases and reverse transcriptases, J. Am. Chem. Soc. 1 19, 3177-3178).

Briefly, PNA is synthesized using chemistries similar to those used for synthesis of nucleic acids and peptides. The PNA monomers used in such syntheses are hybrids of nucleosides and amino acids. The neutral backbone of a PNA oligomer results in unique properties. For example, when PNA is used as a probe for detection of conventional nucleic acids, such properties include: (a) higher affinity; (b) faster hybridization; and (c) relative independence of hybridization from salt concentration.

In general, a given PNA duplexed with a conventional nucleic acid having one or more mismatches will result in a greater change in melting temperature (i.e. ΔT_ΠI). In part due to this property, a wide variety of applications for PNAs have been described (see e.g. Buchardt et al., 1993, TIBTECH 1 1 , 384-386; Orum et al., 1993, Nucleic Acids Research 21, 5332-5336; and Corey et al., 1997, TIBTECH 15, 224-229). PNA products, services, and technical support are available from PerSeptive Biosystems, Inc. (Framingham, Massachusetts; hereinafter "PerSeptive"' www.pbio.com).

Generally, to provide the maximum increase in melting temperature without compromising sequence specificity, it is best to design a PNA having a maximum length of 18 monomer units (from 12 to 15 monomer units is often ideal for most applications). A PNA having the desired sequence of nitrogenous bases may be obtained in several ways. First, PNA may be synthesized using the Expedite™ Nucleic Acid Synthesis system available from PerSeptive. Next, customer PNA synthesis is available from PerSeptive. Finally, PNA oligomers may be manually synthesized using either Fmoc or t-Boc based monomers (available from PerSeptive) and standard peptide chemistry protocols. Standard peptide purification conditions are used to purify PNA following synthesis.

The chemical structure of PNA consists of repeating units of N-(2-aminoethyl)

-glycine linked by amide bonds. The nitrogenous bases are attached to this neutral backbone by methylene carbonyl linkages. Unlike the natural nucleic acid backbone, no deoxyribose or ribose or phosphate groups are present. As a result, as mentioned above, PNA binding to target nucleic acid sequences is stronger than conventional nucleic acid probes, and the binding is virtually independent of salt concentration.

Quantitatively, this is reflected by a higher thermal stability of duplexes containing PNA relative to conventional nucleic acids. For example, a 15 base pair PNA:DNA duplex by about 15°C at 100 mM NaCl (PNA:RNA duplexes have a similar increased T_m). PNA probes therefore bind to DNA or RNA target sequences tightly under a broad range of stringency conditions (i.e. temperature and ionic strength).

PNA has a terminal amine group which may be labeled with all standard labels. Examples of such labels include but are not limited to fluorescein, rhodamine and biotin.

PNA can be used to compete for a primer site in a conventional PCR reaction to improve specificity by a technique known as PCR clamping (see e.g. Orum et al., 1993, Single base pair mutation analysis by PNA directed PCR clamping, Nucl. Acids Res. 21, 5332-5336). Briefly, PCR clamping can be used to resolve single base differences among template strands. In one embodiment, this technique works by competition between a PNA sequence and a DNA primer for a polymoφhic primer site on a template. Here, the PNA prevents amplification of any template having the exact PNA sequence but not the DNA primer sequence. In this way, one can suppress the amplification of a wild type sequence while amplifying a mutant sequence present in the same template reaction mix. In other words, a PNA sequence can be used to selectively suppress amplification of a template molecule having a perfect PNA match while not inhibiting the amplification of a desired sequence that differs by as little as one base. A variety of references describing PCR clamping are available (Thiede et al., 1996, Simple and sensitive detection of mutations in the ras proto-oncogenes using PNA-mediated PCR clamping, Nucl. Acids Res. 24, 983-984; Orum et al., 1997, Peptide Nucleic Acid, in Laboratory Methods for (he Detection of Mutations and Polymorphisms in DNA, Taylor, ed., Chapter 1 1, CRC Press, pp. 123-133; Rhodes et al., 1997, Analysis of the allele specific PCR method for the detection of neoplastic disease, Diag. Mol. Pathol. 6, 49; Mrozikiewicz et al., 1997, Peptide nucleic acid-mediated polymerase chain reaction clamping allows allelic allocation of CYP1A1 Mutations, Anal. Biochem. 250, 256-257; Mrozikiewicz et al., 1997,

CYP1A1 mutations 4887A, 4889G, 5639C and 6235C in the Polish population and their allelic linkage, determined by peptide nucleic acid mediated PCR clamping, Pharmacogenetics 7, 303-307).

Further, a great variety of PNA-based assays for nucleic acid hybridization have been described, the contents of each of which is hereby incoφorated by reference in its entirety (see e.g. Carlsson, et al., 1996, Screening for genetic mutations, Nature 380, 207-207; Rose, 1993, Characterization of antisense binding properties of peptide nucleic acids by capillary gel electrophoresis, Anal. Chem. 65, 3545-3549; Wang et al., 1996, Peptide nucleic acid probes for sequence-specific DNA biosensors, J. Am. Chem. Soc. 1 18, 7667-7670; Orum et al., 1997, Peptide Nucleic Acid, in Laboratory Methods for the Detection of Mutations and Polymorphisms in DNA, Taylor, ed., Chapter 1 1, CRC Press, pp. 123-133; Perry-O'Keefe et al., 1996, PNA pre-gel hybridization, an alternative to southern blotting, Proc. Natl. Acad. Sci. U.S.A. 93, 14670-14675; Hansen et al., 1997 Detection of PNA DNA hybrid molecules by anitobdy Fab fragments isolated from a phage display library, J.

Immunol. Methods 203, 199-207; Weiler et al., 1997, Hybridization based DNA screening on peptide nucleic acid (PNA) oligomer arrays, Nucl. Acids Res. 25, 2792- 2799; Castro and Williams, 1997, Single-molecule detection of specific nucleic acid sequences in unamplified genomic DNA, Anal. Chem. 69, 3915-3920). Extensive additional information on PNA-based assays is available (see e.g. Egholm et al, 1992,

Peptide nucleic acids (PNA): Oligonucleotide analogues with an achiral peptide backbone, J. Am. Chem. Soc. 114, 1895-1897; Egholm et al, 1993, PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen bonding rules, Nature 365, 566-568; Wittung et al., 1994, DNA-like double helix formed by peptide nucleic acid, Nature 368, 561-563; Buchardt et al., 1993, Peptide nucleic acids and their potential applications in biotechnology. TIB TECH 1 1 , 384-386; Orum et al., 1993, Single base pair mutation analysis by PNA directed PCR clamping, Nucl. Acids Res. 21, 5332-5336; Brown et al, 1994, NMR solution structure of a peptide nucleic acid complexed with RNA, Science 265, 777-780; Egholm et al., 1995, Efficient pH-independent sequence-specific DNA binding by pseudoisocytosine- containing bis-PNA, Nucl. Acids Res. 23, 217-222; Orum et al., 1995, Sequence specific purification of nucleic acids by PNA controlled hybrid selection, BioTechniques 19, 472-480; Koch et al., 1995, PNA-Peptide Chimerae, Tetrahed. Lett. 36, 6933-6936; Veselkov et al., 1996, PNA as a rare genome cutter. Nature 379, 214; Norton et al., 1996, Inhibition of human telomerase activity by peptide nucleic acids, Nature Biotechnol. 14, 615-619; Lansdoφ et al., 1996, Heterogeneity in telomer length of human chromosomes, Hum. Mol. Gen. 5, 685-691 ; Corey, 1997, Peptide nucleic acids: expanding the scope of nucleic acid recognition, TIB TECH 15, 224-229; Seeger et al., 1997, PNA-mediated purification of PCR amplifiable human genomic DNA from whole blood, BioTechniques 23, 512-516). Finally, extensive reviews on PNA have been published, the contents of each of which is hereby incorporated by reference in its entirety (Nielsen et al, 1992, Peptide nucleic acids (PNA): Oligonucleotide analogues with a polyamide backbone, in Antisense Research and Applications, Crooke and Lebleu, eds., CRC Press, pp. 363-372; Nielsen et al, 1993, Peptide nucleic acids (PNAs): potential antisense and anti-gene agents, Anti-Cancer Drug Design 8, 53-63; Buchardt et al, 1993, Peptide nucleic acids and their potential applications in biotechnology, TIB TECH 1 1 , 384- 386; Nielsen et al, 1994, Peptide nucleic acid (PNA), a DNA mimic with a peptide backbone, Bioconjugate Chem. 5, 3-7; Nielsen et al., 1996, Peptide nucleic acid (PNA): A lead for gene therapeutic drugs, in Antisense Thereapeutics Vol. 4 (ed.

Trainor, ed., SECOM Science Publishers B.V., Leiden, pp. 76-84: Nielsen, 1995, DNA analogues with nonphosphodiester backbones, Ann. Rev. Biophys. Biomol. Struct. 24, 167-183; Hyrup and Nielsen, 1996, Peptide nucleic acids (PNA): synthesis, properties and potential applications, Bioorg. Med. Chem. 4, 5-23; Mesmaeker et al., 1995, Backbone modifications in oligonucleotides and peptide nucleic acid systems,

Curr. Opin. Struct. Biol. 5, 343-355; Dueholm and Nielsen, 1997, Chemistry, properties, and applications of PNA (peptide nucleic acid), New J. Chem. 21, 19-31 ; Knudsen and Nielsen, 1997, Application of peptide nucleic acid in cancer therapy, Anti-Cancer Drug 8, 113-118; Nielsen, 1997, Design of sequence-specific DNA- binding ligands, Chem. Eur. J. 3, 505-508; Corey, 1997, Peptide nucleic acids: expanding the scope of nucleic acid recognition, TIB TECH 15, 224-229; Nielsen and Orum, 1995, Peptide nucleic acid (PNA), a new molecular tool, in Molecular Biology: Current Innovations and Future Trends, Part 2, Horizon Scientific Press, pp. 73-89; Nielsen and Haaima, 1997, Peptide nucleic acid (PNA), A DNA mimic with a pseudopeptide backbone, Chem. Soc. Rev., 73-78; Orum et al., 1997, Peptide Nucleic

Acid, in Nucleic Acid Amplification Technologies: Application to Disease Diagnostics, Lee et al., eds., BioTechniques Books Div., Eaton Publishing, pp. 29-48).

Antibodies Antibodies of use with the device of the invention include any antibodies known in the art. Such antibodies may be used, for example, to detect a nucleic acid of interest. In this regard, a nucleic acid may be detected by antibody binding to the nucleic acid itself or to an antigen (e.g., a protein, peptide or hapten) which is bound (either covalently or non-covalently) to the nucleic acid. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric and humanized antibodies, as briefly described below. Further, single chain antibodies, Fab fragments and F(ab')₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above may also be used.

Polyclonal antibodies which may be used with the device of the invention are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Various procedures well known in the art may be used for the production of polyclonal antibodies to an antigen-of-intercst. For example, the production of polyclonal antibodies, various host animals can be immunized by injection with an antigen of interest or derivative thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-

Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.

Monoclonal antibodies which may be used with the device of the invention are homogeneous populations of antibodies to a particular antigen. A monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), and the more recent human B cell hybridoma technique (Kozbor et al, 1983, Immunology Today 4, 72), and the EBV-hybridoma technique (Cole et al., 1985,

Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.

Monoclonal antibodies which may be used with the device of the invention include but are not limited to human monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Nat'l Acad. Sci. U.S.A. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; Olsson et al., 1982, Meth. Enzymol. 92, 3-16).

A chimeric antibody may be used with the device of the invention. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murin mAb and a human immunoglobulin constant region. Various techniques are available for the production of such chimeric antibodies (see e.g., Morrison et al, 1984, Proc. Nat'l

Acad. Sci. U.S.A. 81, 6851-6855; Neuberger et al., 1984, Nature, 312, 604-608; Takeda et al., 1985, Nature, 314, 452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity.

A humanized monoclonal antibody may be used with the device of the invention. Briefly, humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Various techniques have been developed for the production of humanized antibodies

(see, e.g., Queen, U.S. Patent No. 5,585,089, which is incoφorated herein by reference in its entirety). An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by tliree hypervariable regions, referred to as complementarity determining regions (CDRs). The extent of the framework region and CDRs have been precisely defined (see, Kabat et al., 1983, Sequences of proteins of immunological interest, U.S. Department of Health and Human services). Alternatively, techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778; Bird, 1988, Science 242, 423-426; Huston et al., 1988, Proc. Nat'l Acad. Sci. U.S.A. 85, 5870-5883; and Ward et al, 1989, Nature 334, 544-546) can be adapted to produce single chain antibodies useful in the device of the invention. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region together via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F

(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246, 1275-1281) to allow rapid and easy identificaiton of monoclonal Fab fragments with the desired specificity.

Further general methods of antibody production and use are suitable for use in connection with the device of the invention. For example see Harlow and Lane, 1988, Antibodies: A Laboratory Manuel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, which is incoφorated herein by reference in its entirety.

The invention described and claimed herein can be further appreciated by one skilled in the art through reference to the examples which follow. These examples are provided merely to illustrate several aspects of the invention and shall not be construed to limit the invention in any way.

Detection of an Organism

The device of the invention may be used, inter alia, to detect an organism, and/or to detect genotype differences among organisms, via detection of a nucleic acid specific to the organism or organisms. Here, any nucleic acid may be used which has a nucleotide sequence known to be specific (i.e. unique ) to a given organism or strain thereof. In a preferred embodiment, detection is facilitated through the use of amplified DNA. DNA may be amplified using any technique known in the art (e.g. bacterial plasmid replication, polymerase chain reaction).

For example, the determination of Mycobacteria (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansasii) and strains thereof may be accomplished through the amplification of specific regions of genomic DNA by PCR. The requirements allowing distinction among DNA templates are two-fold, as set forth below.

First, a PCR product to be analyzed is denatured. This may be performed using any method known to one skilled in the art. For example, the following three methods are suitable: (a) the TargEx™ method; (b) denaturation by heat (see e.g. Bawa et al., 1995, Indian J. Med. Res. 101 , 142-146) and/or chemical means; and (c) using Competitive Binding Oligonucleotides (CBOs), as described below.

The TargEx™ (Molecular Tool, Baltimore, Maryland) method for generating a single-stranded template from a PCR product consists of adding T7 gene 6 exonuclease (e.g. 50 U/μl from USB Amersham, Arlington Heights, Illinois) directly to a PCR reaction. The method works by using a set of primers in which one of the two primers is fluorescein labeled for signal detection and has four phosphorothioate bases at the 5' end to protect the strand from digestion by the T7 gene 6 exonuclease. The exonuclease is added to the PCR reaction to a final concentration of 0.6 U/μl and the mixture is incubated for one hour at room temperature to generate the single- stranded template.

The denaturation method for generating a single-stranded template from a PCR product consists of heating the PCR product above its denaturing temperature and bringing it into contact with specific probes before re-annealing can fully occur. Alternatively, the PCR product may be kept in a denatured state following heating by using chemical denaturants (e.g. formamide). The CBO method for generating a single-stranded template from a PCR product consists of using a CBO (i.e. a DNA, PNA, RNA, or other modified oligonucleotide) that can hybridize to the PCR template at one or more regions other than where the PCR primers or the specific probes hybridize, such that when the PCR product is heat denatured and allowed to re-anneal in the presence of the CBOs, the CBOs will hybridize to the PCR template at the CBO target regions, thereby preventing complete re-annealing of the PCR product strands. The CBO method allows an area to be designated as a capture region by the lack of the presence of a CBO, thus allowing the specific probe to hybridize during the assay.

The second requirement to allow distinction among DNA templates derived from different organisms or strains thereof is detectably labeling such templates. Here, the PCR product itself, or a secondarily-hybridizable element or covalently- attachable element, may be labeled to allow the detection of said PCR product. This can be accomplished by any number of standard immunological and/or biochemical processes known in the art. Such processes include but are not limited to biotinylated PCR primers, fluorescemated secondary capture oligonucleotides, digoxigenin-labeled anti-double strand DNA antibodies, and the like. The result is a PCR product which can be detected using standard immunological or biochemical techniques. In a preferred embodiment, the method used with the device of the invention employs biotinylated PCR primers.

Hybridization may be carried out in any suitable hybridization buffer. In one embodiment, a suitable buffer comprises 1.5 M NaCl and 10 mM EDTA. In a preferred embodiment, hybridization is carried out in the presence of GBA hybridization buffer (see Nikiforov et al., 1994, Genetic bit analysis: a solid phase method for typing single nucleotide polymoφhisms, Nucl. Acids Res. 22, 4167- 4175). The GBA hybridization buffer may be combined with the PCR solution in a liquid or dry state. Visualization of a PCR product hybridized to a specific probe may be accomplished using any method known in the art. In one embodiment, visualization is carried out using biotinylated primers and colored, streptavidin-labeled microparticles. In a preferred embodiment, such microparticles are dark blue, 0.35 μm polystyrene microparticles. Alternatively, visualization may be carried out using biotinylated primers and a streptavidin/alkaline phosphatase conjugate. Here, the visual signal is developed in the presence of a color-generating alkaline phosphatase substrate (e.g. BCIP/NBT solution, Sigma Chemical Company, St. Louis, Missouri).

Any number of organisms may be distinguished from one another using the device of the invention and known detection means. Sub-types of a particular organism (i.e. strains, varieties, sub-species, etc.) may also be distinguished. The use of detection means which are well-known in the art (i.e. nucleic acid probes) together with the device of the invention permits detection of virtually all known organisms for which a unique nucleic acid probe sequence is available.

For example, a class of enterotoxic E. coli, 0157:H7, may be distinguished from other organisms and from other E. coli using known nucleic acid probes and the device of the invention. Further examples or organisms which may be detected using known detection means and the device of the invention include but are not limited to Chlamydia,

Gonorrhoeae, Staphylococcus, Candida, Hepatitis viruses, HIV, etc. (see e.g. Beristain et al., 1995, Evaluation of a dipstick method for the detection oϊ human immunodeficiency virus infection, J. Clin. Lab. Anal. 9, 347-350; O'Brien et al, 1988, Use of a leukocyte esterase dipstick to detect Chlamydia trachomatis and Neisseria gonorrhoea urethritis in asymptomatic adolescent male detainees, Am. J. Pub. Health 78,

1583-1584; Morissette et al., 1991, Rapid and sensitive sandwich enzyme-linked immunosorbent assay for detection of Staphylococcal enterotoxin B in cheese, Appl. Environ. Microbiol. 57, 836-842; Weissberg, 1978, Evaluation of a dipstick for Candida, Obstet. Gynecol. 52, 506-509; Kim an Doyle, 1992, Dipstick immunoassay to detect enterohemorrhagic Escherichia coli 0157:H7 in retail ground beef, Appl. Environ.

Microbiol. 58, 1764-1767; Mulyanto et al., 1996, An easy dipstick assay for anti-core antibodies to screen blood donors for hepatitis C virus viremia, Vox Sanguinis 70, 229- 231). Any amplified DNA target derived from an organism can be detected and distinguished with this device.

Detection of a Genotype

The genotype of an organism maybe determined using the device of the invention together with any of the many genotyping methods known in the art. Techniques similar to those outlined above for detection of an organism or strains thereof may be used. Additional methods will be apparent to one skilled in the art.

For example, genotype data can be determined using any number of common immunochemical techniques in conjunction with standard protocols. This may involve the use of a streptavidin/alkaline phosphatase conjugate to detect the presence of a biotinylated nucleic acid which was extended from the 3' end of one or more specific capture probes.

Any number of genes can be distinguished or typed with the use of this device. For example, the device can be configured for detection of HLA-DR class II antigens. In one embodiment, a single biotin-labeled PCR product may be produced and detected (see e.g. Abe et al., 1992, Rapid DNA typing utilizing immobilized oligonucleotide probe and a nonradioactive detection system, J. Immunol. Meth. 154, 205-210). In another embodiment, the device may be configured with probes specific for distinct HLA-DRB genotypes (see e.g. Table 1 of Abe et al., Id.). Thus, a labeled PCR product and its corresponding genotypes may be distinguished using the device of the invention and known detection means which have been incorporated into the device as pre-deposited reagents (see also Vaughan, 1991, PCR-SSO typing for HLA-DRB alleles, Eur. J. Immunogenet. 18, 69-80; Erlich et al., 1991, HLA-DR, DQ and DP typing using PCR amplification and immobilized probes, Eur. J. Immunogent. 18, 33-55; and Buyse et al, 1993, Rapid DNA typing of class II HLA antigens using the polymerase chain reaction and reverse dot blot hybridization, Tissue Antigens 41, 1 -14). The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed since these embodiments are intended as illustration of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Throughout this application various references are cited, the contents of each of which is hereby incorporated by reference into the present application in its entirety.

Claims

WE CLAIM:

1 . A device for detection of a nucleic acid in a sample from a subject comprising: a reaction chamber having reagents pre-deposited therein for preparation of the sample; and a moveable membrane situated within the reaction chamber having reagents pre-deposited thereon for processing of the sample.

2. The device of Claim 1 , wherein the reaction chamber comprises a flattened window area suitable for viewing the moveable membrane within.

3. The device of Claim 1 , wherein the reaction chamber is attached to a fluid transfer tube.

4. The device of Claim 3, wherein the fluid transfer tube comprises a standpipe for preventing the sample from being ejected during positioning of the moveable membrane.

5. The device of Claim 4, wherein the fluid transfer tube is attached to a micro- well adaptor.

6. The device of Claim 5, wherein the micro-well adaptor is attached to an aspiration tube.

7. The device of Claim 6, wherein the aspiration tube is of a suitable diameter to fit within a capillary polymerase chain reaction tube.

8. The device of Claim 6, wherein the aspiration tube has an outer diameter of from 200 micrometers to 1 millimeter.

9. The device of Claim 1 , wherein the nucleic acid is amplified prior to detection by an amplification method.

10. The device of Claim 9, wherein the amplification method is polymerase chain reaction, ligase amplification reaction or rolling circle amplification.

1 1. The device of Claim 1 , wherein detection of the nucleic acid in the sample is indicative of the presence of a disease, disorder, or organism in the subject.

12. The device of Claim 1 1 , wherein the organism is selected from the group consisting of herpesvirus, Neisseria gonorrhea, human immunodeficiency virus, Epstein Barr virus, Helicobacter pylori, Haemophilus influenzae, and Mycoplasma genitalium.

13. The device of Claim 1 , wherein the subject is a plant or an animal.

14. The device of Claim 1 , wherein the moveable membrane having reagents pre- deposited thereon comprises one or more immobilized probes.

15. The device of Claim 14, wherein the moveable membrane further comprises one or more probe markers for visibly marking the position of the one or more immobilized probes.

16. A device for detection of a nucleic acid in a sample from a subject comprising: a reaction chamber having reagents pre-deposited therein for preparation of the sample; a membrane situated within the reaction chamber having reagents pre- deposited thereon for processing of the sample; and a membrane positioner for changing the position of the membrane within the reaction chamber.

17. The device of Claim 1 or Claim 16, wherein the membrane comprises a material selected from the group consisting of nylon, nitrocellulose, paper, plastic and polyethersulfone.

18. The device of Claim 16, wherein the membrane positioner is a plunger or a screw.

19. The device of Claim 16, wherein the reaction chamber comprises threads capable of threading the membrane positioner.

20. The device of Claim 16, wherein the membrane positoner positions the membrane by pushing on an absorbent material attached to the top of the membrane.

21. The device of Claim 16, wherein the reaction chamber is a barrel or tube.

22. The device of Claim 21 , wherein the barrel is a 1 ml, 3 ml, 5 ml or 10 ml syringe barrel.

23. A device for detection of a nucleic acid in a sample from a subject comprising: a reaction chamber having reagents pre-deposited therein for preparation of the sample; a fluid transfer tube attached to the floor of the reaction chamber for transferring the sample into the reaction chamber; a membrane situated within the reaction chamber having reagents pre- deposited thereon for processing of the sample; and a membrane positioner for dipping the membrane into the sample following sample preparation.

24. The device of Claim 23, wherein a micro-well PCR device adaptor is attached to the fluid transfer tube.

25. The device of Claim 24, wherein an aspiration tube is attached to the micro- well PCR device adaptor.

26. The device of Claim 23, wherein the membrane is constructed from a material selected from the group consisting of nitrocellulose, nylon, paper, plastic and polyethersulfone.

27. The device of Claim 23 having a plurality of probes immobilized on the membrane, wherein one or more of the plurality is capable of hybridizing under conditions of high stringency with the nucleic acid in the sample.

28. The device of Claim 27 having a plurality of probe markers selected from the group consisting of inks, dyes and stains visibly marking the position on the membrane of one or more of the plurality of probes.

29. The device of Claim 23, wherein a pre-deposited reagent carrier is situated on the membrane and comprises a polyfiberglass pad containing a signaling or detection reagent.

30. The device of Claim 29, wherein the signaling or detection reagent comprises 0.01 to 100 μm colored beads coated with avidin or streptavidin.

31. The device of Claim 23, wherein the nucleic acid is labeled with biotin.

32. The device of Claim 23, wherein a positive control site for color control is located on the membrane and comprises a biotinylated molecule.

33. The device of Claim 23, wherein the reaction chamber is constructed from a material selected from the group consisting of polypropylene, polyethylene, polycarbonate, metal and glass.

34. The device of Claim 23, wherein the fluid transfer tube is constructed from a material selected from the group consisting of polypropylene, polyethylene, polycarbonate, polyimide, metal and glass.

35. The device of Claim 23, wherein a micro-well PCR device adaptor is attached to the fluid transfer tube and is constructed from a material selected from the group consisting of polypropylene, polyethylene, polycarbonate, metal and glass.

36. The device of Claim 35, wherein an aspiration tube is attached to the micro- well PCR device adaptor and is constructed from a material selected from the group consisting of polypropylene, polyethylene, polycarbonate, polyimide, metal and glass.

37. The device of Claim 23, wherein the reagents pre-deposited in the reaction chamber comprise one or more components suitable for adjustment of hybridization stringency.

38. The device of Claim 23, wherein the membrane positioner comprises a pressure regulator for aspiration of the sample into the reaction chamber.

39. The device of Claim 23, wherein the membrane positioner for dipping the membrane into the sample is selected from the group consisting of a plunger and a screw.

40. The device of Claim 23, wherein the reagents pre-deposited on the membrane comprise a microparticle selected from the group consisting of plastic, latex, carbon, magnetite and gold.

41. The device of Claim 40, wherein the microparticle ranges from 10 nm to 500 μm diameter.

42. The device of Claim 40, wherein the microparticle is labeled with a colored dye or a fluorescent label.

43. The device of Claim 40, wherein the microparticle is labeled with a protein or a nucleic acid.

44. The device of Claim 43, wherein the nucleic acid is selected from the group consisting of deoxyribonucleic acid, ribonucleic acid and protein nucleic acid.

45. The device of Claim 23, wherein an absorbent material selected from the group consisting of cotton, felt, paper, polyfiberglass and wool is situated at the end of the membrane opposite the dipping end.

46. The device of any one of Claims 1 , 16 and 23, wherein the membrane further comprises a pre-deposited reagent carrier having signaling or detection reagents pre- deposited thereon.

47. The device of Claim 46, wherein the pre-deposited reagent carrier is positioned on the membrane so as to be dipped into the sample following sample preparation.

48. The device of Claim 37, wherein one or more components suitable for adjustment of hybridization stringency is selected from the group consisting of inorganic salts, formamide and detergents.

49. The device of Claim 23, wherein the fluid transfer tube attached to the floor of the reaction chamber comprises a standpipe for preventing the sample from being ejected during positioning of the membrane.

50. The device of Claim 23, wherein the membrane comprises a plurality of probes immobilized thereon, a plurality of probe markers visibly marking the position of each of the plurality of probes, and a positive control site.

51. The device of Claim 38, wherein the pressure regulator is constructed from a material selected from the group consisting of rubber and silicone.

52. The device of Claim 43, wherein the protein is selected from the group consisting of an enzyme, an antibody, a fluorescent protein, avidin, NeutrAvidin and streptavidin.

53. The device of Claim 11, wherein the disease or disorder is selected from the group consisting of tuberculosis, gonorrhea, acquired immunodeficiency syndrome and cardiovascular disease.