WO2013105981A1 - Procédés et dispositifs de détection et d'identification rapides de microorganismes vivants par des aptamères et/ou des anticorps immobilisés sur des membranes perméables - Google Patents

Procédés et dispositifs de détection et d'identification rapides de microorganismes vivants par des aptamères et/ou des anticorps immobilisés sur des membranes perméables Download PDF

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Publication number
WO2013105981A1
WO2013105981A1 PCT/US2012/022739 US2012022739W WO2013105981A1 WO 2013105981 A1 WO2013105981 A1 WO 2013105981A1 US 2012022739 W US2012022739 W US 2012022739W WO 2013105981 A1 WO2013105981 A1 WO 2013105981A1
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Prior art keywords
permeable membrane
target microorganism
antibody
aptamer
group
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PCT/US2012/022739
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English (en)
Inventor
Bret T. BARNHIZER
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Nanologix, Inc.
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Priority claimed from US13/350,127 external-priority patent/US9068216B2/en
Application filed by Nanologix, Inc. filed Critical Nanologix, Inc.
Priority to BR112014017336A priority Critical patent/BR112014017336A8/pt
Priority to PCT/US2013/021482 priority patent/WO2013106853A1/fr
Publication of WO2013105981A1 publication Critical patent/WO2013105981A1/fr
Priority to IN6784DEN2014 priority patent/IN2014DN06784A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes

Definitions

  • the present invention relates generally to the field of microbiology and, in particular, to microbiological diagnostics for the rapid detection and identification of target microorganisms.
  • Modern microbiological diagnostic assays employ two different growth protocols for analysis, i.e., detection, identification and/or enumeration, of microorganisms: (1) analysis without preliminary growth of the microorganisms or (2) analysis after preliminary growth of microorganisms. Analysis of microorganisms without preliminary growth includes the use of methods such as: (1) immunological analyses, e.g., immunofluorescence, radioimmunoassay, enzyme immunoassay (EIA) for single cells and others; (2) DNA/RNA analyses via polymerase chain reaction (PCR); and (3) flow cytometry (FC) analyses (detection of single cells after labeling with fluorescent antibodies or fluorogenic substrates).
  • immunological analyses e.g., immunofluorescence, radioimmunoassay, enzyme immunoassay (EIA) for single cells and others
  • EIA enzyme immunoassay
  • PCR polymerase chain reaction
  • FC flow cytometry
  • Multibiological diagnostic assays that analyze microorganisms after preliminary growth of the microorganisms include enzyme-linked immunosorbent assay (ELISA), mass-spectrometry, Fourier transform infrared (FTIR) spectroscopy, immune analyses and others.
  • ELISA enzyme-linked immunosorbent assay
  • FTIR Fourier transform infrared
  • Colonies appearing on solid nutrient media typically are counted for detection and enumeration of total microbial growth or are removed and analyzed according to traditional microbiological procedures, e. g., mass-spectrometry, Fourier transform infrared spectroscopy (FTIR) spectroscopy, chromatography, immunoassays or PCR.
  • FTIR Fourier transform infrared spectroscopy
  • Lactobacillus spp. Bacillus cereus, some yeast and molds and other microorganisms.
  • Pathogens which require rapid identification for military and civilian defense include, for example, anthrax, where time is of the essence.
  • the present invention fulfills this need by providing methods and devices for rapid detection and identification of live target microorganisms contained in a liquid sample or after a short growth time of a sample on nutrient media.
  • the detection and identification of target microorganisms may be achieved in as little as one hour or less, a very short time period not previously encountered with other diagnostic assays known to date.
  • a method for rapid detection and identification of one or more live target microorganisms in a sample comprising the steps of obtaining a sample containing at least one target microorganism; forming a mixture containing the at least one microcolony from the at least one target microorganism and at least one first aptamer conjugated to a reporter compound and selected for a binding site of the at least one target microorganism and/ or of at least one first antibody conjugated to a reporter compound and specific for an antigenic site of the at least one target microorganism; washing the mixture; adding the mixture to a permeable membrane, the permeable membrane having immobilized thereon at least one second aptamer selected for a binding site of the at least one first aptamer and/ or at least one second antibody specific for an antigenic site of the at least one first antibody; washing any non-bound microorganisms from the permeable membrane; and adding a detection solution to the permeable membrane in
  • the sample is a liquid sample containing at least one or more live target microorganisms.
  • the sample is obtained by placing a sample containing at least one target microorganism on a nutrient medium and incubating the sample for a period of time in order to grow at least one microcolony from the at least one target microorganism.
  • a method for rapid detection and identification of one or more live target microorganisms in a sample comprising the steps of obtaining a sample containing at least one target microorganism; forming a mixture containing the at least one microcolony from the at least one target microorganism and at least one first aptamer conjugated to a reporter compound and selected for a first binding site of the at least one target microorganism and/ or at least one first antibody conjugated to a reporter compound and specific for a first antigenic site of the at least one target microorganism; adding the mixture to a permeable membrane having immobilized thereon at least one second aptamer selected for a second binding site of the at least one target microorganism and/or at least one second antibody specific for a second antigenic site of the at least one target microorganism; washing any non-bound microorganisms from the permeable membrane; and adding a detection solution to the permeable membrane in order to detect and
  • the sample is a liquid sample containing at least one or more live target microorganisms.
  • the sample is placed on a nutrient medium and incubated for a period of time in order to grow at least one microcolony from the at least one target microorganism.
  • a device for rapid detection and identification of one or more live target microorganisms in a sample comprising a container having therein nutrient medium to grow at least one microcolony from at least one live target microorganism; at least one first aptamer conjugated to a reporter compound and selected against a binding site of the at least one target microorganism and/or at least one first antibody conjugated to a reporter compound and specific for an antigenic site of the at least one target microorganism; a permeable membrane having immobilized thereon at least one second aptamer selected against a binding site of the at least one first aptamer and/or at least one second antibody specific for an antigenic site of the at least one first antibody; a portable washing apparatus; and a detection solution for detecting and identifying the at least one target microorganism bound on the permeable membrane in one hour or less.
  • a device for rapid detection and identification of one or more live target microorganisms in a sample comprising a container having therein nutrient medium to grow at least one microcolony from at least one live target microorganism; at least one first aptamer conjugated to a reporter compound and selected against a first binding site of the at least one target microorganism and/or at least one first antibody conjugated to a reporter compound and specific for a first antigenic site of the at least one target microorganism; a permeable membrane having immobilized thereon at least one second aptamer selected against a second binding site of the at least one target microorganism and/or at least one second antibody specific for a second antigenic site of the at least one target microorganism; a portable washing apparatus; and a detection solution for detecting and identifying the at least one target microorganism bound on the permeable membrane in one hour or less.
  • the invention also includes test kits comprised of the methods and devices described hereinabove.
  • the at least one second aptamer of the invention may be linked to an amine compound, which includes without limitation, bovine serum albumin, amine linkers and other compounds which contain an amine group.
  • the at least one type of antibody includes, without limitation, a monoclonal or a polyclonal antibody.
  • Target microorganisms that can be detected and identified according to the embodiments of the present invention include, without limitation, bacteria, yeasts, fungi, viruses, cells and eukaryotic cells.
  • Suitable nutrient media contemplated for growing the at least one microorganism in a sample include, without limitation, agar such as tryptic soy agar or sheep blood agar.
  • Suitable permeable membranes contemplated for use in the present invention include, without limitation, polymers such as nitrocellulose, regenerated cellulose and its derivatives such as cellophane, cuprophane and dialysis membranes, nylon or polyvinylidene fluoride (PVDF) and other polymers such as organic or inorganic materials with naturally occurring pores or artificially created pores.
  • polymers such as nitrocellulose, regenerated cellulose and its derivatives such as cellophane, cuprophane and dialysis membranes, nylon or polyvinylidene fluoride (PVDF) and other polymers such as organic or inorganic materials with naturally occurring pores or artificially created pores.
  • PVDF polyvinylidene fluoride
  • Suitable reporter compounds contemplated for use in the present invention include, without limitation, chromogenic substrates such as tetrazolium dyes such as MTT [3-(4,5-dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide], INT [2-(p-iodophenyl-3-(p-nitrophenyl tetrazolium chloride], resazurine and other chromogenic substrates; fluorogenic substrates such as fluorescein isothiocyanate (FITC); and enzymes such as horse-radish peroxidase.
  • chromogenic substrates such as tetrazolium dyes such as MTT [3-(4,5-dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide], INT [2-(p-iodophenyl-3-(p-nitrophenyl tetrazolium chloride], resazurine and other chromogenic substrate
  • Suitable detection solutions contemplated for use in the present invention include, without limitation, enzymes such as alkaline phosphatase and staining solutions such as 3,3'-diaminobenzidine (DAB).
  • enzymes such as alkaline phosphatase
  • staining solutions such as 3,3'-diaminobenzidine (DAB).
  • DAB 3,3'-diaminobenzidine
  • Portable washing apparatuses are well known in the art, any of which may be used in the methods and devices of the invention.
  • the methods and devices of the invention allow for rapid detection and identification of target microorganisms in one hour or less.
  • target microorganisms of the invention are detected and identified in 30 minutes or less.
  • Fig. 1 shows an aptamer (referred to herein as a first aptamer) bound by another aptamer (referred to herein as a second aptamer).
  • the first aptamer is conjugated to a reporter compound and is bound to a target microorganism.
  • the second aptamer is immobilized on a permeable membrane via linkage to an amine compound.
  • Fig. 2 shows an aptamer (referred to herein as a first aptamer) bound to one site of a target microorganism, the first aptamer conjugated to a reporter compound, and a permeable membrane having an immobilized aptamer thereon (referred to herein as a second aptamer) bound to a second site of the target microorganism.
  • the second aptamer is linked to an amine compound which immobilizes the second aptamer to the permeable membrane.
  • Fig. 3 shows an antibody (referred to herein as a first antibody) bound by another antibody (referred to herein as a second antibody).
  • the first antibody is conjugated to a reporter compound and is bound to a target microorganism.
  • the second antibody is immobilized on a permeable membrane.
  • Fig. 4 shows an antibody (referred to herein as a first antibody) bound to one site of a target microorganism, the first antibody conjugated to a reporter compound, and a permeable membrane having an immobilized antibody thereon (referred to herein as a second antibody), the second antibody bound to a second site of the target microorganism.
  • a first antibody an antibody bound to one site of a target microorganism
  • the first antibody conjugated to a reporter compound and a permeable membrane having an immobilized antibody thereon (referred to herein as a second antibody)
  • a permeable membrane having an immobilized antibody thereon referred to herein as a second antibody
  • Fig. 5 shows an antibody (referred to herein as a first antibody) bound by an aptamer (referred to herein as a second aptamer), the first antibody conjugated to a reporter compound and bound to a target microorganism.
  • the second aptamer is immobilized on a permeable membrane via linkage to an amine compound.
  • Fig. 6 shows an antibody (referred to herein as a first antibody) bound to one site of a target microorganism, the first antibody conjugated to a reporter compound, and a permeable membrane having an immobilized aptamer thereon (referred to herein as a second aptamer).
  • the second aptamer is bound to a second site of the target microorganism and is immobilized on the permeable membrane via linkage to an amine compound.
  • Fig. 7 shows an aptamer (referred to herein as a first aptamer) bound by an antibody (referred to herein as a second antibody), the first aptamer conjugated to a reporter compound and bound to a target microorganism.
  • the second antibody is immobilized on a permeable membrane.
  • Fig. 8 shows an aptamer (referred to herein as a first aptamer) bound to one site of a target microorganism, the first aptamer conjugated to a reporter compound, and a permeable membrane having an immobilized antibody thereon (referred to herein as a second antibody).
  • the second antibody is bound to a second site of the target microorganism.
  • the present invention provides methods and devices for detecting and identifying live microorganisms which are extremely rapid and which do not require sophisticated laboratory equipment such as flow cytometers, mass-spectrometers, FTIR spectroscopy or PCR.
  • the present invention is based on the ability of live target microorganisms to bind selectively and with high specificity to unbound aptamers and or antibodies and to aptamers and/or antibodies immobilized on permeable membranes.
  • the present invention allows for the detection and identification of many different types of microorganisms, such as bacteria, yeasts, fungi, spores, eukaryotic cells, cells, viruses or proteins.
  • Aptamers nucleic acid sequences having unique properties, were first identified from very large random sequence oligonucleotide libraries in 1990 by Tuerk C. et al. (Science, 1990, 249:505-10) and Ellington, A.D. et al. (Nature, 1990; 346: 818-22), both papers incorporated herein by reference.
  • the selection method for aptamers called the SELEX (systemic evolution of ligands by exponential enrichment) process, is a technique which screens very large combinatorial libraries of oligonucleotides by an iterative process of in vitro selection and amplification.
  • Combinatorial libraries based on replicable biopolymers such as nucleic acids (and peptides) offer the convenience of iterative amplification of their members, making the screening process fast and easy.
  • a random sequence oligonucleotide library is incubated with a target of interest.
  • a very small fraction of individual sequences tends to interact with the target, and these sequences are separated from the rest of the library by means of any one of several physical separation techniques known in the art.
  • nitrocellulose filter partitioning is used with protein targets that are retained on nitrocellulose.
  • Small molecular targets are generally immobilized on a solid support to generate an affinity matrix, in which case sequences that do not interact with the target on the solid support can be removed easily by a simple washing step.
  • the population of sequences bound to the target is isolated and amplified to obtain an enriched library to be used for the next
  • the enrichment efficiency of high-affinity binders is governed by the stringency of selection at each round. The progress of the enrichment of high-affinity binders can be determined by carrying out binding analysis of enriching populations against the target. Once affinity saturation is achieved after several rounds of selection/ amplification, the enriched library is cloned and sequenced to obtain the sequence information of each member. Individual sequences are further characterized on the basis of their ability to bind to the target. The majority of individual sequences, greater than 90%, in an enriched library are aptamers that bind successfully to the target used for selection.
  • Aptamers have the capacity to recognize virtually any class of target compounds with high affinity and specificity which rival antibodies in both therapeutic and diagnostic applications. Aptamers are much smaller and less complex than antibodies, and thus are easier to manufacture and modify. Aptamers mimic properties of antibodies in a variety of diagnostic formats, but are very different from antibodies, having characteristics that overcome many shortcomings of antibodies when used in diagnostic applications.
  • Some of the shortcomings of antibodies include difficulty in growing high yields of monoclonal antibodies in vivo, as they typically are grown as hybridomas in peritoneal cavities of animals and purified from ascites fluid; performance of the same antibody tends to vary from batch to batch, requiring immunoassays to be re-optimized with each new batch of antibodies; identification of antibodies that could recognize targets under conditions other than physiological conditions is not feasible; kinetic parameters of antibody-target interactions cannot be changed on demand; antibodies are sensitive to temperature and undergo irreversible denaturation; and antibodies have a limited shelf life.
  • aptamers possess many advantages when used in diagnostic applications.
  • the aptamer selected against theophylline, a purine derivative used to treat asthma binds with a 10,000-fold lower affinity to caffeine, another purine analog, which differs from theophylline by only one methyl group.
  • Third, selection conditions can be manipulated to obtain aptamers with properties desirable for in vitro diagnostics.
  • aptamers that bind to a target in a non-physiological buffer and at non-physiological temperatures can be identified.
  • kinetic parameters such as the on- and off-rates of aptamers, can be changed on demand.
  • animals or cells are not involved in aptamer identification, toxins as well as compounds which do not elicit good immune responses can be used to generate high-affinity aptamers.
  • aptamers are produced by chemical synthesis with extreme accuracy and reproducibility. They are purified under denaturing conditions to a very high degree of purity. Therefore, little or no batch-to-batch variation is encountered in aptamer production.
  • reporter compounds such as biotin, HRP and fluorescein can be attached to aptamers at precise locations identified by the user.
  • Functional groups that allow subsequent derivatization of aptamers with other compounds can also be attached during chemical synthesis of aptamers.
  • Seventh although aptamers undergo denaturation, the process is reversible. After being denatured, functional aptamers can be regenerated easily within minutes. Eighth, aptamers are stable to long-term storage and can be transported at ambient temperature, and thus can tolerate transportation without special requirements for cooling, eliminating the need for a continuous cold chain.
  • the present invention takes advantage of the superior qualities and unique characteristics of aptamers in concert with antibody technology to arrive at extremely rapid assays to detect and identify target microorganisms in as little one hour or less and, in most cases, 30 minutes or less.
  • the permeable membranes encompassed by the present invention include, without limitation, polymers such as nitrocellulose, regenerated cellulose and its derivatives such as cellophane, cuprophane and dialysis membranes, nylon or polyvinylidene fluoride (PVDF) and other polymers such as organic or inorganic materials with naturally occurring pores or artificially created pores.
  • nitrocellulose membranes are able to bind amine (NH 2 )-containing compounds on their surfaces because of the strong interaction of nitrite (N0 2 ) groups contained in the nitrocellulose membrane with the NH 2 groups contained in the compounds.
  • the aptamer compounds encompassed by the present invention are linked to NH 2 -containing compounds which include, without limitation, amine linkers, biotin, bovine serum albumin (BS A) compounds or other NH 2 -containing compounds, and thus are easily immobilized on the permeable membranes of the present invention.
  • NH 2 -containing compounds include, without limitation, amine linkers, biotin, bovine serum albumin (BS A) compounds or other NH 2 -containing compounds, and thus are easily immobilized on the permeable membranes of the present invention.
  • the NH 2 -containing compounds are linked to the 5'-end of the aptamer compounds during oligonucleotide synthesis, according to synthetic protocols known by those in the art. All antibody compounds contain NH 2 groups, and thus the antibodies encompassed by the present invention also are easily immobilized on permeable membranes.
  • Monoclonal antibodies, polyclonal antibodies or a combination of both may be used in the methods of the present invention.
  • Lectins or other natural agglutinins also may be used in place of antibodies.
  • a blocking buffer compound such as milk powder, BSA, biotin or other proteinaceous compounds, so that all spaces of the permeable membrane are filled.
  • a permeable membrane 10 is shown with an aptamer 18 (referred to herein as a second aptamer) immobilized thereon.
  • the second aptamer 18 is linked to an amine compound 40 which immobilizes the second aptamer 18 to the permeable membrane 10.
  • the second aptamer 18 is bound to a different aptamer 14 (referred to herein as a first aptamer), which is bound to a target microorganism 32.
  • the first aptamer 14 is conjugated to a reporter compound 30 which includes, without limitation, chromogenic substrates such as tetrazolium dyes such as MTT [3-(4,5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], INT [2-(p-iodophenyl-3-(p-nitrophenyl tetrazolium chloride], resazurine and other chromogenic substrates; fluorogenic substrates such as fluorescein isothiocyanate (FITC); or enzymes such as horse-radish peroxidase.
  • chromogenic substrates such as tetrazolium dyes such as MTT [3-(4,5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], INT [2-(p-iodophenyl-3-(p-nitrophenyl tetrazolium chloride], resazurine and other chrom
  • a detection solution which includes, without limitation, 3,3'-diaminobenzidine (DAB) or alkaline phosphatase, is added to the permeable membrane 10 which changes the color of the permeable membrane 10 in locations where the target microorganism 32 is attached to the permeable membrane 10 via binding to the immobilized second aptamer 18.
  • DAB 3,3'-diaminobenzidine
  • alkaline phosphatase alkaline phosphatase
  • a permeable membrane 10 is shown with an aptamer 18 (referred to herein as a second aptamer) immobilized thereon.
  • the second aptamer 18 is linked to an amine compound which immobilizes the second aptamer 18 to the permeable membrane 10.
  • the second aptamer 18 is bound to one site of a target microorganism 32 and a different aptamer 14 (referred to herein as a first aptamer) is bound to a different site of the target microorganism 32.
  • the first aptamer 14 is conjugated to a reporter compound 30 as described above.
  • a detection solution as described above, is added to the permeable membrane 10 which changes the color of the permeable membrane 10 in locations where the target microorganism 32 is attached to the permeable membrane 10 via binding to the immobilized second aptamer 18.
  • a permeable membrane 10 is shown with an antibody 26 (referred to herein as a second antibody) immobilized thereon.
  • the second antibody 26 is bound to a different antibody 22 (referred to herein as a first antibody), which is bound to a target microorganism 32.
  • the first antibody 22 is conjugated to a reporter compound 30 as described above.
  • a detection solution as described above, is added to the permeable membrane 10 which changes the color of the permeable membrane 10 in locations where the target microorganism 32 is attached to the permeable membrane 10 via binding to the immobilized second antibody 26.
  • a permeable membrane 10 is shown with an antibody 26 (referred to herein as a second antibody) immobilized thereon.
  • the second antibody 26 is bound to an antigenic site of a target microorganism 32 and a different antibody 22 (referred to herein as a first antibody) is bound to a second antigenic site of the target microorganism 32.
  • the first antibody 22 is conjugated to a reporter compound 30 as described above.
  • a detection solution as described above, is added to the permeable membrane 10 which changes the color of the permeable membrane 10 in locations where the target microorganism 32 is attached to the permeable membrane 10 via binding to the immobilized antibody 26.
  • a permeable membrane 10 is shown with an aptamer 18 (referred to herein as a second aptamer) immobilized thereon.
  • the aptamer 18 is linked to an amine compound 40 which immobilizes the second aptamer 18 to the permeable membrane 10.
  • the second aptamer 18 is bound to an antibody 22 (referred to herein as a first antibody), which is bound to a target microorganism 32.
  • the first antibody 22 is conjugated to a reporter compound as described above.
  • a detection solution as described above, is added to the permeable membrane 10 which changes the color of the permeable membrane 10 in locations where the target microorganism 32 is attached to the permeable membrane 10 via binding to the immobilized second aptamer 18.
  • a permeable membrane 10 is shown with an aptamer 18 (referred to herein as a second aptamer) immobilized thereon.
  • the second aptamer 18 is linked to an amine compound which immobilizes the second aptamer 18 to the permeable membrane 10.
  • the second aptamer 18 is bound to one site of a target microorganism 32 and an antibody 22 (referred to herein as a first antibody) is bound to a different antigenic site of the target microorganism 32.
  • the first antibody 22 is conjugated to a reporter compound 30 as described above.
  • a detection solution as described above, is added to the permeable membrane 10 which changes the color of the permeable membrane 10 in locations where the target microorganism 32 is attached to the permeable membrane 10 via binding to the immobilized second aptamer 18.
  • a permeable membrane 10 is shown with an antibody 26 (referred to herein as a second antibody) immobilized thereon.
  • the second antibody 26 is bound to an aptamer 14 (referred to herein as a first aptamer), which is bound to a target microorganism 32.
  • the first aptamer 14 is conjugated to a reporter compound as described above.
  • a detection solution as described above, is added to the permeable membrane 10 which changes the color of the permeable membrane 10 in locations where the target microorganism 32 is attached to the permeable membrane 10 via binding to the immobilized second antibody 26.
  • a permeable membrane 10 is shown with an antibody 26 (referred to herein as a second antibody) immobilized thereon.
  • the second antibody 26 is bound to an antigenic site of a target microorganism 32 and an aptamer 14 (referred to herein as a first aptamer) is bound to a different site of the target microorganism 32.
  • the first aptamer 14 is conjugated to a reporter compound 30 as described above.
  • a detection solution as described above, is added to the membrane 10 which changes the color of the permeable membrane 10 in locations where the target microorganism 32 is attached to the permeable membrane 10 via binding to the immobilized second antibody 26.
  • the present invention encompasses not only the foregoing described configurations of aptamers and/or antibodies, but also encompasses having more than one type of aptamer and/or more than one type of antibody immobilized on the permeable membrane and more than one type of aptamer and/or more than one type of antibody conjugated to a reporter compound, in order to allow for rapid detection and identification of more than one target microorganism.
  • a liquid sample containing an unknown number of live bacterial species including the target bacterium GBS (also known as Streptococcus agalactiae) is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the target bacterium GBS is poured on a Petri plate filled with sheep blood agar (SBA); the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • SBA sheep blood agar
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml. To each dilution, a 1 :30 dilution of horse-radish peroxidase (HRP) conjugated to aptamers selected against GBS is added to bring the final volume up to 100 ml. The sample is spun at 3,000 rpm in a
  • phosphate buffered saline PBS
  • the pellet is resuspended in PBS.
  • Twenty ml of the resuspended sample is added to a nitrocellulose permeable membrane which has been blocked overnight with 1% bovine serum albumin blocking compounds and containing immobilized aptamers (linked via amine compounds to the membrane) selected to bind to the aptamers conjugated with HRP.
  • the permeable membrane then is washed three times.
  • a drop of 3,3'- diaminobenzidine (DAB) (3 mg/ml in Tris saline buffer, pH 7.6), 3% H 2 0 2 and 1.2 weight % of NiCl 2 then is added to the permeable membrane.
  • DAB 3,3'- diaminobenzidine
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where GBS is attached to the membrane via binding to the immobilized aptamers.
  • GBS then is detected and identified on the permeable membrane due to the color change of the GBS microcolony(ies), which is distinguished from the color of any other species of bacteria contained in the mixture.
  • GBS is detected reliably at 10 5 to 10 2 bacteria/ ml in as little as 30 minutes, with minimal background observed, and with no cross-reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the target bacterium GBS is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the target bacterium GBS is poured on a Petri plate filled with SBA; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to aptamers selected against one antigenic site of GBS is added to bring the final volume up to 20 ml.
  • the 20 ml sample is added to a nitrocellulose permeable membrane which has been blocked overnight with 1% BSA blocking compounds and containing immobilized aptamers (linked via amine compounds to the membrane) selected to bind to a second antigenic site of GBS.
  • the permeable membrane then is washed in a washing solution (PBS, pH 7.4 with 0.1 % Tween 20) using a portable washing device at medium speed of rotation for about two minutes.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where GBS is attached to the membrane via binding to the immobilized aptamers.
  • GBS then is detected and identified on the permeable membrane due to the color change of the GBS microcolony(ies), which is distinguished from the color of any other species of bacteria contained in the mixture.
  • GBS is detected reliably at 10 5 to 10 2 bacteria/ml in as little as 30 minutes, with minimal background observed, and with no cross-reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the target bacterium GBS is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the target bacterium GBS is poured on a Petri plate filled with SBA; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to rabbit polyclonal antibodies against GBS is added to bring the final volume up to 100 ml.
  • the sample is spun at 3,000 rpm in a microcentrifuge and washed with PBS three times.
  • the pellet is resuspended in PBS.
  • Twenty ml of the resuspended sample is added to a nitrocellulose permeable membrane which has been coated with 20 ml of a 1:40 dilution of mouse monoclonal antibody against rabbit IgG and blocked overnight with 1% BS A blocking compounds.
  • the permeable membrane then is washed three times.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where GBS is attached to the membrane via binding to the immobilized antibodies.
  • GBS then is detected and identified on the permeable membrane due to the color change of the GBS microcolony(ies), which is distinguished from the color of any other species of bacteria contained in the mixture.
  • GBS is detected reliably at 10 5 to 10 2 bacteria/ml in as little as 30 minutes, with minimal background observed, and with no cross-reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the target bacterium GBS is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the target bacterium GBS is poured on a Petri plate filled with SBA; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to rabbit polyclonal antibodies specific for an antigenic site of GBS is added to bring the final volume up to 20 ml.
  • the 20 ml sample is added to a nitrocellulose permeable membrane which has been coated with 20 ml of a 1 :40 dilution of rabbit polyclonal antibodies specific for a different antigenic site of GBS and blocked overnight with 1% BSA blocking compounds.
  • the permeable membrane then is washed in a washing solution (PBS, pH 7.4 with 0.1 % Tween 20) using a portable washing device at medium speed of rotation for about two minutes.
  • a liquid sample containing an unknown number of live bacterial species including the target bacterium anthrax is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the target bacterium anthrax is poured on a Petri plate filled with SB A; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to aptamers selected against anthrax is added to bring the final volume up to 100 ml.
  • the sample is spun at 3,000 rpm in a microcentrifuge and washed with PBS three times.
  • the pellet is resuspended in PBS.
  • Twenty ml of the resuspended sample is added to a nitrocellulose permeable membrane which has been blocked overnight with 1% BSA blocking compounds and containing immobilized aptamers (linked to amine linker compounds) selected to bind to the aptamers conjugated with HRP.
  • the permeable membrane then is washed three times.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where anthrax is attached to the membrane via binding to the immobilized aptamers.
  • Anthrax then is detected and identified on the permeable membrane due to the color change of the anthrax microcolony(ies), which is distinguished from the color of any other species of bacteria contained in the mixture.
  • Anthrax is detected reliably at 10 5 to 10 2 bacteria ml in as little as 30 minutes, with minimal background observed, and with no cross-reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the target bacterium anthrax is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the target bacterium anthrax is poured on a Petri plate filled with SB A; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to aptamers selected against one antigenic site of anthrax is added to bring the final volume up to 20 ml.
  • the 20 ml sample is added to a nitrocellulose permeable membrane which has been blocked overnight with 1 % BSA blocking compounds and containing immobilized aptamers (linked via amine compounds to the membrane) selected to bind to a second antigenic site of anthrax.
  • the permeable membrane then is washed in a washing solution (PBS, pH 7.4 with 0.1 % Tween 20) using a portable washing device at medium speed of rotation for about two minutes.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where anthrax is attached to the membrane via binding to the immobilized aptamers.
  • Anthrax then is detected and identified on the permeable membrane due to the color change of the anthrax microcolony(ies), which is distinguished from the color of any other species of bacteria contained in the mixture.
  • Anthrax is detected reliably at 10 5 to 10 2 bacteria/ml in as little as 30 minutes, with minimal background observed, and with no cross-reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the target bacterium anthrax is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the target bacterium GBS is poured on a Petri plate filled with SB A; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to rabbit polyclonal antibodies against anthrax is added to bring the final volume up to 100 ml.
  • the sample is spun at 3,000 rpm in a microcentrifuge and washed with PBS three times. The pellet is resuspended in PBS. Twenty ml of the resuspended sample is added to a nitrocellulose permeable membrane which has been coated with 20 ml of a 1:40 dilution of mouse monoclonal antibody against rabbit IgG and blocked overnight with 1% BSA blocking compounds. The permeable membrane then is washed three times.
  • a liquid sample containing an unknown number of live bacterial species including the target bacterium anthrax is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the target bacterium anthrax is poured on a Petri plate filled with SB A; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to rabbit polyclonal antibodies specific for an antigenic site of anthrax is added to bring the final volume up to 20 ml.
  • the 20 ml sample is added to a nitrocellulose permeable membrane which has been coated with 20 ml of a 1 :40 dilution of rabbit polyclonal antibodies specific for a different antigenic site of anthrax and blocked overnight with 1 % BSA blocking compounds.
  • the permeable membrane then is washed in a washing solution (PBS, pH 7.4 with 0.1 % Tween 20) using a portable washing device at medium speed of rotation for about two minutes.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where anthrax is attached to the membrane via binding to the immobilized antibodies.
  • Anthrax then is detected and identified on the permeable membrane due to the color change of the anthrax microcolony(ies), which is distinguished from the color of any other species of bacteria contained in the mixture.
  • Anthrax is detected reliably at 10 5 to 10 2 bacteria/ml in as little as 30 minutes, with minimal background observed, and with no cross-reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the two target bacteria E. coli and Salmonella is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the two target bacteria E. coli and Salmonella are poured on a Petri plate filled with SBA; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to aptamers selected against E. coli and aptamers selected against Salmonella is added to bring the final volume up to 100 ml.
  • the sample is spun at 3,000 rpm in a microcentrifuge and washed with PBS three times. The pellet is resuspended in PBS. Twenty ml of the resuspended sample is added to a nitrocellulose permeable membrane which has been blocked overnight with 1 % BSA blocking compounds and containing immobilized thereon two different aptamers (each linked via amine compounds to the membrane).
  • the first aptamer is selected to bind to the aptamers conjugated with HRP and selected against E.
  • the second aptamer is selected to bind to the aptamers conjugated with HRP and selected against Salmonella.
  • the two different aptamers are localized on the permeable membrane in distinct locations to distinguish between the two species of bacteria.
  • the permeable membrane then is washed three times.
  • a drop of DAB (3 mg/ml in Tris saline buffer, pH 7.6), 3% H 2 0 2 and 1.2 weight % of N1CI 2 then is added to the permeable membrane.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where E. coli and Salmonella are attached to the membrane via binding to their respective immobilized aptamers.
  • E. coli and Salmonella then are detected and identified on the permeable membrane due to the color change of the E. coli and Salmonella on the permeable membrane and their respective locations on the permeable membrane, which is distinguished from the color of any other species of bacteria which may be contained in the mixture.
  • E. coli and Salmonella are detected reliably at 10 5 to 10 2 bacteria/ml in as little as 30 minutes, with minimal background observed, and with no cross-reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the two target bacteria E. coli and Salmonella is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the two target bacteria E. coli and Salmonella are poured on a Petri plate filled with SBA; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to two different aptamers one aptamer selected for a first antigenic site of E. coli and the other aptamer selected for a first antigenic site of Salmonella, is added to bring the final volume up to 20 ml.
  • the 20 ml sample is added to a nitrocellulose permeable membrane which has been blocked overnight with 1 % BSA blocking compounds and which has been coated with 20 ml of a 1 :40 dilution of two aptamers (each linked via an amine compound to the membrane), the first aptamer selected for a second antigenic site of E. coli and the second aptamer selected for a second antigenic site of Salmonella.
  • the permeable membrane then is washed in a washing solution (PBS, pH 7.4 with 0.1 % Tween 20) using a portable washing device at medium speed of rotation for about two minutes.
  • a drop of DAB (3 mg/ml in Tris saline buffer, pH 7.6), 3% H 2 O 2 and 1.2 weight % of N1CI 2 then is added to the permeable membrane.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where E. coli and Salmonella are attached to the membrane via binding to their respective immobilized aptamers. E. coli and Salmonella then are detected and identified on the permeable membrane due to the color change of the E.
  • E. coli and Salmonella on the permeable membrane and their respective locations on the permeable membrane, which is distinguished from the color of any other species of bacteria which may be contained in the mixture.
  • E. coli and Salmonella are detected reliably at 10 5 to 10 2 bacteria/ml in as little as 30 minutes, with minimal background observed, and with no cross-reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the two target bacteria E. coli and Salmonella is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the two target bacteria E. coli and Salmonella are poured on a Petri plate filled with SBA; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to antibodies specific for an antigenic site of E. coli and antibodies specific for an antigenic site of Salmonella is added to bring the final volume up to 100 ml.
  • the sample is spun at 3,000 rpm in a microcentrifuge and washed with PBS three times. The pellet is resuspended in PBS. Twenty ml of the resuspended sample is added to a nitrocellulose permeable membrane which has been blocked overnight with 1 % BSA blocking compounds and containing immobilized thereon two different antibodies.
  • the first antibody is specific to an antigenic site of the antibodies conjugated with HRP and specific to an antigenic site of E.
  • the second antibody is specific to an antigenic site of the antibodies conjugated with HRP and specific to an antigenic site of Salmonella.
  • the two different antibodies are localized on the permeable membrane in distinct locations to distinguish between the two species of bacteria.
  • the permeable membrane then is washed three times.
  • a drop of DAB (3 mg/ml in Tris saline buffer, pH 7.6), 3% 3 ⁇ 4(1 ⁇ 4 and 1.2 weight % of N1CI 2 then is added to the permeable membrane.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where E. coli and Salmonella are attached to the membrane via binding to their respective immobilized antibodies.
  • E. coli and Salmonella then are detected and identified on the permeable membrane due to the color change of the E. coli and Salmonella on the permeable membrane and their respective locations on the permeable membrane, which is distinguished from the color of any other species of bacteria which may be contained in the mixture.
  • E. coli and Salmonella are detected reliably at 10 5 to 10 2 bacteria/ml in as little as 30 minutes, with minimal background observed, and with no cross-reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the two target bacteria E. coli and Salmonella is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the two target bacteria E. coli and Salmonella are poured on a Petri plate filled with SBA; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to two different rabbit polyclonal antibodies one antibody specific for a first antigenic site of E. coli and the other antibody specific for a first antigenic site of Salmonella, is added to bring the final volume up to 20 ml.
  • the 20 ml sample is added to a nitrocellulose permeable membrane which has been blocked overnight with 1 % BSA blocking compounds and which has been coated with 20 ml of a 1 :40 dilution of two rabbit polyclonal antibodies, the first antibody specific for a second antigenic site of E. coli and the second antibody specific for a second antigenic site of Salmonella.
  • the permeable membrane then is washed in a washing solution (PBS, pH 7.4 with 0.1 % Tween 20) using a portable washing device at medium speed of rotation for about two minutes.
  • a drop of DAB (3 mg/ml in Tris saline buffer, pH 7.6), 3% H 2 0 2 and 1.2 weight % of NiCl 2 then is added to the permeable membrane.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where E. coli and Salmonella are attached to the membrane via binding to their respective immobilized antibodies. E. coli and Salmonella then are detected and identified on the permeable membrane due to the color change of the E.
  • E. coli and Salmonella on the permeable membrane and their respective locations on the permeable membrane, which is distinguished from the color of any other species of bacteria which may be contained in the mixture.
  • E. coli and Salmonella are detected reliably at 10 5 to 10 2 bacteria/ml in as little as 30 minutes, with minimal background observed, and with no cross-reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the two target bacteria E. coli and Salmonella is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the two target bacteria E. coli and Salmonella are poured on a Petri plate filled with SBA; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to aptamers selected against E. coli and antibodies specific for an antigenic site of Salmonella is added to bring the final volume up to 100 ml.
  • the sample is spun at 3,000 rpm in a microcentrifuge and washed with PBS three times. The pellet is resuspended in PBS. Twenty ml of the resuspended sample is added to a nitrocellulose permeable membrane which has been blocked overnight with 1% BS A blocking compounds and containing immobilized thereon two different aptamers (each linked to amine compounds). The first aptamer is selected to bind to the aptamer conjugated with HRP and selected against E.
  • the second aptamer is selected to bind to the antibodies conjugated with HRP and specific for an antigenic site of Salmonella.
  • the two different aptamers are localized on the permeable membrane in distinct locations to distinguish between the two species of bacteria.
  • the permeable membrane then is washed three times.
  • a drop of DAB (3 mg/ml in Tris saline buffer, pH 7.6), 3% H 2 O 2 and 1.2 weight % of N1CI 2 then is added to the permeable membrane.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where E. coli and Salmonella are attached to the membrane via binding to their respective immobilized aptamers.
  • E. coli and Salmonella then are detected and identified on the permeable membrane due to the color change of the E. coli and Salmonella on the permeable membrane and their respective locations on the permeable membrane, which is distinguished from the color of any other species of bacteria which may be contained in the mixture.
  • E. coli and Salmonella are detected reliably at 10 5 to 10 2 bacteria/ml in as little as 30 minutes, with minimal background observed, and with no cross- reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the two target bacteria E. coli and Salmonella is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the two target bacteria E. coli and Salmonella are poured on a Petri plate filled with SBA; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to an aptamer selected for a first antigenic site of E. coli and an antibody specific for a first antigenic site of Salmonella is added to bring the final volume up to 20 ml.
  • the 20 ml sample is added to a nitrocellulose permeable membrane which has been blocked overnight with 1 % BSA blocking compounds and which has been coated with 20 ml of a 1 :40 dilution of two aptamers (each linked via an amine compound to the membrane), the first aptamer selected for a second antigenic site of E. coli and the second aptamer selected for a second antigenic site of Salmonella.
  • the permeable membrane then is washed in a washing solution (PBS, pH 7.4 with 0.1 % Tween 20) using a portable washing device at medium speed of rotation for about two minutes.
  • a drop of DAB (3 mg/ml in Tris saline buffer, pH 7.6), 3% H 2 O 2 and 1.2 weight % of N1CI 2 then is added to the permeable membrane.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where E. coli and Salmonella are attached to the membrane via binding to their respective immobilized aptamers. E. coli and Salmonella then are detected and identified on the permeable membrane due to the color change of the E.
  • E. coli and Salmonella on the permeable membrane and their respective locations on the permeable membrane, which is distinguished from the color of any other species of bacteria which may be contained in the mixture.
  • E. coli and Salmonella are detected reliably at 10 5 to 10 2 bacteria/ml in as little as 30 minutes, with minimal background observed, and with no cross- reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the two target bacteria E. coli and Salmonella is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the two target bacteria E. coli and Salmonella are poured on a Petri plate filled with SBA; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to aptamers selected against E. coli and antibodies specific for an antigenic site of Salmonella is added to bring the final volume up to 100 ml.
  • the sample is spun at 3,000 rpm in a microcentrifuge and washed with PBS three times. The pellet is resuspended in PBS. Twenty ml of the resuspended sample is added to a nitrocellulose permeable membrane which has been blocked overnight with 1 % BSA blocking compounds and containing immobilized thereon two different antibodies. The first antibody is selected to bind to the aptamers conjugated with HRP and selected against E.
  • the second antibody is selected to bind to the antibodies conjugated with HRP and specific for an antigenic site of Salmonella.
  • the two different antibodies are localized on the permeable membrane in distinct locations to distinguish between the two species of bacteria.
  • the permeable membrane then is washed three times.
  • a drop of DAB (3 mg/ml in Tris saline buffer, pH 7.6), 3% H 2 0 2 and 1.2 weight % of NiCl 2 then is added to the permeable membrane.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where E. coli and Salmonella are attached to the membrane via binding to their respective immobilized antibodies.
  • E. coli and Salmonella then are detected and identified on the permeable membrane due to the color change of the E. coli and Salmonella on the permeable membrane and their respective locations on the permeable membrane, which is distinguished from the color of any other species of bacteria which may be contained in the mixture.
  • E. coli and Salmonella are detected reliably at 10 5 to 10 2 bacteria/ml in as little as 30 minutes, with minimal background observed, and with no cross-reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.
  • a liquid sample containing an unknown number of live bacterial species including the two target bacteria E. coli and Salmonella is set up as described below.
  • a sample containing an unknown mixture of live bacterial species including the two target bacteria E. coli and Salmonella are poured on a Petri plate filled with SBA; the plate is incubated at a temperature of about 37° C for about 3 to 6 hours to grow the bacteria; and, after the bacterial species is grown and microcolonies appear, a sample specimen of the microcolonies is set up as described below.
  • a 0.5 McFarland of the liquid sample or, alternatively, a sample of the grown microcolonies, is prepared as commonly known in the art and the bacteria are serially diluted, starting at about 10 5 bacteria/ml.
  • a 1 :30 dilution of HRP conjugated to an aptamer selected for a first antigenic site of E. coli and an antibody specific for a first antigenic site of Salmonella is added to bring the final volume up to 20 ml.
  • the 20 ml sample is added to a nitrocellulose permeable membrane which has been blocked overnight with 1 % BSA blocking compounds and which has been coated with 20 ml of a 1 :40 dilution of two antibodies, the first antibody specific for a second antigenic site of E. coli and the second antibody specific for a second antigenic site of Salmonella.
  • the permeable membrane then is washed in a washing solution (PBS, pH 7.4 with 0.1 % Tween 20) using a portable washing device at medium speed of rotation for about two minutes.
  • a drop of DAB (3 mg/ml in Tris saline buffer, pH 7.6), 3% H 2 0 2 and 1.2 weight % of NiCl 2 then is added to the permeable membrane.
  • a color reaction to a grey-brown color rapidly develops in about 0.5 to 3 minutes on the surface of the permeable membrane in locations where E. coli and Salmonella are attached to the membrane via binding to their respective immobilized antibodies. E. coli and Salmonella then are detected and identified on the permeable membrane due to the color change of the E.
  • E. coli and Salmonella on the permeable membrane and their respective locations on the permeable membrane, which is distinguished from the color of any other species of bacteria which may be contained in the mixture.
  • E. coli and Salmonella are detected reliably at 10 5 to 10 2 bacteria/ml in as little as 30 minutes, with minimal background observed, and with no cross-reactivity to other species of bacteria. This detection and identification assay is significantly more rapid than any other microbiological diagnostic assay known to date.

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Abstract

L'invention concerne des procédés, des dispositifs et des kits permettant de détecter et d'identifier rapidement un ou plusieurs microorganismes cibles vivants dans un échantillon liquide ou ayant poussé sur des plaques contenant un milieu nutritif solide. L'invention consiste à mélanger un ou plusieurs microorganismes cibles avec un ou plusieurs aptamères et/ou un ou plusieurs anticorps chacun conjugués à un composé rapporteur et spécifique à un premier site sur un ou plusieurs microorganismes cibles afin de former un mélange. Le mélange est placé sur une membrane perméable sur laquelle sont immobilisés un ou plusieurs aptamères liés à un composé amine et/ou un ou plusieurs anticorps, chacun spécifique à un second site sur un ou plusieurs microorganismes cibles ou un site sur le conjugué d'aptamère et/ou le conjugué d'anticorps. Une solution de détection est ajoutée à la membrane, et la détection et l'identification d'un ou de plusieurs microorganismes cibles se fait en une heure environ ou moins.
PCT/US2012/022739 2012-01-13 2012-01-26 Procédés et dispositifs de détection et d'identification rapides de microorganismes vivants par des aptamères et/ou des anticorps immobilisés sur des membranes perméables WO2013105981A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
BR112014017336A BR112014017336A8 (pt) 2012-01-13 2012-01-26 método para rápida detecção e identificação de um ou mais microorganismos alvo vivos em uma amostra; dispositivo para rápida detecção e identificação de um ou mais microorganismos alvo vivos em uma amostra; e teste de kit para rápida detecção e identificação de um ou mais microorganismos alvo vivos em uma amostra para uso no método
PCT/US2013/021482 WO2013106853A1 (fr) 2012-01-13 2013-01-14 Procédés et dispositifs de détection et d'identification rapides de micro-organismes vivants ou de leurs protéines sécrétées sur des membranes perméables par fixation à des aptamères et/ou des anticorps
IN6784DEN2014 IN2014DN06784A (fr) 2012-01-13 2014-08-12

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13/350,127 2012-01-13
US13/350,127 US9068216B2 (en) 2007-03-22 2012-01-13 Methods and devices for rapid detection and identification of live microorganisms by aptamers and/or antibodies immobilized on permeable membranes

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WO2013105981A1 true WO2013105981A1 (fr) 2013-07-18

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PCT/US2013/021482 WO2013106853A1 (fr) 2012-01-13 2013-01-14 Procédés et dispositifs de détection et d'identification rapides de micro-organismes vivants ou de leurs protéines sécrétées sur des membranes perméables par fixation à des aptamères et/ou des anticorps

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PCT/US2013/021482 WO2013106853A1 (fr) 2012-01-13 2013-01-14 Procédés et dispositifs de détection et d'identification rapides de micro-organismes vivants ou de leurs protéines sécrétées sur des membranes perméables par fixation à des aptamères et/ou des anticorps

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020240570A1 (fr) * 2019-05-28 2020-12-03 Ramot At Tel-Aviv University Ltd. Systèmes et procédés de détection d'un organisme pathogène

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US6503702B1 (en) * 1993-12-10 2003-01-07 Syngenta Investment Corporation Rapid immunoassay for detection of antibodies or antigens incorporating simultaneous sample extraction and immunogenic reaction
US20100227338A1 (en) * 2007-03-22 2010-09-09 Nanologix, Inc. Method and Apparatus for Rapid Detection and Identification of Live Microorganisms Immobilized On Permeable Membrane by Antibodies

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6696254B2 (en) * 2001-11-21 2004-02-24 Kimberly-Clark Worldwide, Inc. Detection and identification of enteric bacteria
WO2008118400A1 (fr) * 2007-03-22 2008-10-02 Nanologix, Inc. Détection et identification de microorganismes sur des membranes perméables transparentes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6503702B1 (en) * 1993-12-10 2003-01-07 Syngenta Investment Corporation Rapid immunoassay for detection of antibodies or antigens incorporating simultaneous sample extraction and immunogenic reaction
US20100227338A1 (en) * 2007-03-22 2010-09-09 Nanologix, Inc. Method and Apparatus for Rapid Detection and Identification of Live Microorganisms Immobilized On Permeable Membrane by Antibodies

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020240570A1 (fr) * 2019-05-28 2020-12-03 Ramot At Tel-Aviv University Ltd. Systèmes et procédés de détection d'un organisme pathogène
CN114402195A (zh) * 2019-05-28 2022-04-26 拉莫特特拉维夫大学有限公司 用于侦测一病原体生物的数个系统及数个方法

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BR112014017336A2 (pt) 2017-06-13
BR112014017336A8 (pt) 2017-07-04
WO2013106853A1 (fr) 2013-07-18

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