METHODS AND DEVICES FOR THE SELECTIVE DETECTION OF MICROORGANISMS
Methods and devices are provided for the rapid and specific detection of target microorganisms, cells, and the like. In one embodiment, the methods involve contacting a target microorganism (e.g., in a sample) with a permeabilization reagent that selectively permeabilizes or lyses the microorganism; contacting the selectively permeabilized microorganism with a detection reagent that is taken into the selectively permeabilized organism or that contacts metabolites or enzymes released by the selectively permeabilized microorganism, where the detection reagent produces a signal in the presence of said metabolites or enzymes; and detecting a signal produced by the detection reagent in the presence of the metabolites or enzymes wherein the strength of the signal indicates the presence and/or amount of the target microorganism in the sample.
1 . A method of detecting or quantifying a target microorganism in a sample, said method comprising:
contacting said target microorganism with a selective permeabilization reagent that selectively permeabilizes or lyses said microorganism;
contacting the selectively permeabilized microorganism with a detection reagent that is taken into said selectively permeabilized organism or that contacts metabolites or enzymes released by said selectively permeabilized microorganism, wherein said detection reagent produces a signal in the presence of said metabolites or enzymes; and
detecting a signal produced by said detection reagent in the presence of said metabolites or enzymes wherein the strength of said signal indicates the presence and/or amount of said target microorganism in said sample.
2 . The method of claim 1 , wherein said metabolites or enzymes comprise a metabolite or enzyme selected from the group consisting of ATP, DNA, RNA, calcium, beta-galactosidase (beta-gal), beta-glucuronidase, alcohol dehydrogenase or other NAD oxidoreductase, a transferase, an alkaline phosphatase or other hydrolase, a lyase, an isomerase, an oxidase, a gyrase, a DNA nuclease (DNases), and 1RNA nuclease (RNase), and a restriction enzyme.
3 . The method of claim 2 , wherein said metabolites or enzymes comprise ATP.
4 . The method of claim 3 , wherein said detection reagent comprises a luciferase and said signal comprises a luminescence signal.
5 . The method of claim 3 , wherein said detection reagent comprises a target responsive electrochemical aptamer switch (TREAS) for ATP detection and said signal comprises an electrochemical signal.
6 . The method of claim 3 , wherein said detection reagent comprises a molecular beacon (MB)-like DNA for the detection of ATP and said signal comprises a fluorescent signal.
7 . The method of claim 3 , wherein said detection reagent comprises an enzyme substrate and said detecting comprises detecting a reaction between the released enzyme and said enzyme substrate.
8 . The method of claim 7 , wherein said enzyme substrate is a substrate for an enzyme selected from the group consisting of beta-galactosidase (beta-gal), beta-glucuronidase, alcohol dehydrogenase or other NAD oxidoreductases, transferases, alkaline phosphatases or other hydrolases, lyases, isomerases, oxidases, gyrases, a DNA nuclease (DNases), and 1RNA nuclease (RNase), and a restriction enzyme.
9 . The method of claim 8 , wherein said substrate is selected from the group consisting of coumarin-4-acetic acid 7-O-caprylate, coumarin-4-acetic acid 7-O-beta-D-glucuronide, and coumarin-4-acetic acid 7-O-beta-D-galactopyranoside.
10 . The method of claim 3 , wherein said detection reagent comprises an enzyme and a substrate for that enzyme and said detecting comprise detecting the reaction between the enzyme and the substrate in the presence of a cofactor or a coenzyme that is released from said microorganism.
11 . The method of claim 10 , wherein said detection reagent comprises an enzyme that uses NAD, NADP, or FAD as a cofactor.
12 . The method of claim 7 , wherein said enzyme substrate and/or said enzyme is provided on and/or in a solid support.
13 . The method of claim 12 , wherein said substrate comprises glucose or another substrate for glucose oxidase, and glucose dehydrogenase.
14 . The method of claim 13 , wherein said detecting comprises detecting the reduction of one or more coenzymes selected from the group consisting of NAD, NADP, and FAD.
15 . The method of claim 12 , wherein said substrate comprises hexokinase, a hexose, glucose-6-phosphate dehydrogenase, and NAD.
16 . The method of claim 15 , wherein said detecting comprises detecting released ATP by detecting the reduction of said NAD to NADH.
17 . The method of claim 12 , wherein said substrate comprises glucose-6-phosphate dehydrogenase.
18 . The method of claim 17 , wherein said detecting comprises detecting released NAD by detecting the reduction of said NAD to NADH.
19 . The method of claim 12 , wherein the detection of the reduction of NAD NADP, or FAD comprises detection of a colorimetric reagent that changes color when oxidized or reduced.
20 . The method of claim 12 , wherein the detection of the reduction of NAD NADP or FAD comprises electrochemical detection of a reagent that is oxidized or reduced.
21 . The method of claim 12 , wherein said substrate comprises a test strip compatible with a glucometer readout device.
22 . (canceled)
23 . The method of claim 12 , wherein said contacting the target microorganism with a selective permeabilization reagent occurs on and/or in said substrate.
24 . The method of claim 12 , wherein said contacting the target microorganism with a selective permeabilization reagent occurs in a sample collection device before application to said substrate.
25 . The method of claim 1 , wherein said permeabilization reagent comprises a reagent that disrupts or permeabilizes a microorganism or cell attached to a targeting peptide or antibody that preferentially or specifically binds to said target microorganism.
26 . (canceled)
27 . The method of claim 25 , wherein said targeting peptide is a targeting peptide selected from the targeting peptides listed in Table 2.
28 . The method of claim 25 , wherein said targeting peptide is attached directly or indirectly to an antimicrobial peptide.
29 . The method of claim 28 , wherein said antimicrobial peptide is an antimicrobial peptide selected from the antimicrobial peptides listed in Table 4.
30 . The method of claim 25 , wherein said target microorganism is S. mutans , and said targeting peptide attached to an antimicrobial peptide comprises an amino acid sequence selected from the group consisting of TFFRLFNRSFTQALGKGGGKNLRIIRKGIHIIKKY (C16G2, SEQ ID NO:1110), KFINGVLSQFVLERKPYPKLFKFLRKHLL (1845L621, SEQ ID NO:1111), FIDSFIRSFGGGKLFKFLRKHLL (b43BD2.21, (SEQ ID NO:1112), TFFRLFNRSFTQALGKGGGFLKFLKKFFKKLKY (C16AF5, (SEQ ID NO:1113), FIKHFIHRFGGGKNLRIIRKGIHIIKKY (2 — 1G2, (SEQ ID NO:1115), and KKHRKHRKHRKH GGSGGS KNLRRIIRKGIHIIKKYG (G10KHc, (SEQ ID NO:1115).
31 - 33 . (canceled)
34 . The method of claim 1 , wherein said sample comprises a sample from saliva, plaque, urine, feces, cerebrospinal fluid, blood, vaginal secretions, soil, a surface swab, an agricultural product, a meat product, a poultry product, and a fish product.
35 . A method of detecting or quantifying a target microorganism in a sample, said method comprising:
contacting said target microorganism with a permeabilization reagent that selectively permeabilizes said microorganism;
contacting the selectively permeabilized microorganism with a cell-impermeant label; and
detecting said label in said cell where the presence or amount of said label associated with a microorganism indicates the presence or amount of said target microorganism in said sample.
36 . The method of claim 35 , wherein said detecting comprises a method selected from the group consisting of microscopy, flow cytometry, solid phase cytometry, luminometry, and spectroscopy.
37 . The method of claim 35 , wherein said impermeant label comprises a label selected from the group consisting of propidium iodide, SYTOX Green, SYBR®-14, YoYo®-1, YO-PRO™-1, BO-PRO-1, PO-PRO-1, YO-PRO-1, TO-PRO-1, TO-PRO-3, BO-PRO-3, YO-PRO-3, TO-PRO-#, POPO-1, BOBO-1, YOYO-1, TOTO-1, POPO-3, BOBO-2, YOYO-3, TOTO-3, ethidium homodimers-1, ethidium homodimers-2, ethidium bromide, ethidium monoazide, and Trypan blue.
38 - 39 . (canceled)
40 . The method of claim 35 , wherein said permeabilization reagent comprises a reagent that disrupts or permeabilizes a microorganism attached to a targeting peptide that preferentially or specifically binds to said target microorganism.
41 - 44 . (canceled)
45 . The method of claim 40 , wherein said target microorganism is S. mutans , and said permeabilization reagent comprises an amino acid sequence selected from the group consisting of TFFRLFNRSFTQALGK GGG KNLRIIRKGIHIIKKY (C16G2, SEQ ID NO:1110), KFINGVLSQFVLERK PYP KLFKFLRKHLL (1845L621, SEQ ID NO:1111), FIDSFIRSF GGG KLFKFLRKHLL (b43BD2.21, (SEQ ID NO:1112), TFFRLFNRSFTQALGK GGG FLKFLKKFFKKLKY (C16AF5, (SEQ ID NO:1113), FIKHFIHRF GGG KNLRIIRKGIHIIKKY (2 — 1G2, (SEQ ID NO:1114), and KKHRKHRKHRKH GGSGGS KNLRRIIRKGIHIIKKYG (G10KHc, (SEQ ID NO:1115).
46 - 49 . (canceled)
50 . A diagnostic test device, said device comprising:
a substrate test strip comprising a selective permeabilization reagent;
an enzyme substrate; and
a detection reagent that detects a change in oxidation state of a coenzyme.
51 . The device of claim 50 , wherein said substrate comprises glucose or another substrate for glucose oxidase, and glucose dehydrogenase.
52 . The device of claim 51 , wherein said substrate comprises one or more coenzymes selected from the group consisting of NAD and FAD.
53 . The device of claim 50 , wherein said substrate comprises hexokinase, a hexose, glucose-6-phosphate dehydrogenase, and NAD.
54 - 55 . (canceled)
56 . The device of claim 50 , wherein the detection reagent that is detectable using an electrochemical detection device.
57 - 58 . (canceled)
59 . A diagnostic test unit comprising:
a swab member carried by a housing base defining a sample chamber;
a housing cap comprising a first reagent chamber wherein said housing cap interfits with said housing base to cooperatively form a capped sample chamber with said swab disposed therein and a break-off nib, channel, or port that communicates between said first reagent chamber and said sample chamber; and
a permeabilization reagent that selectively permeabilizes or lyses a target microorganism wherein said permeabilization reagent is disposed within said first reagent chamber or within said sample chamber.
60 - 68 . (canceled)
69 . The diagnostic test unit of claim 59 , wherein said target microorganism is S. mutans , and said targeting peptide attached to an antimicrobial peptide comprises an amino acid sequence selected from the group consisting of TFFRLFNRSFTQALGKGGGKNLRIIRKGIHIIKKY (C16G2, SEQ ID NO:1110), KFINGVLSQFVLERKPYPKLFKFLRKHLL (1845L621, SEQ ID NO:1111), FIDSFIRSFGGGKLFKFLRKHLL (b43BD2.21, (SEQ ID NO:1112), TFFRLFNRSFTQALGKGGGFLKFLKKFFKKLKY (C16AF5, (SEQ ID NO:1113), and FIKHFIHRFGGGKNLRIIRKGIHIIKKY (2 — 1G2, (SEQ ID NO:1114), and KKHRKHRKHRKH GGSGGS KNLRRIIRKGIHIIKKYG (G10KHc, (SEQ ID NO:1115).
70 . (canceled)