Fluorescent assay for bacterial gram reaction
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/04
C12Q-001/68
G01N-033/00
C07H-001/00
출원번호
US-0146328
(1993-11-01)
발명자
/ 주소
Roth Bruce L. (Corvallis OR) Millard Paul J. (Eugene OR) Yue Stephen T. (Eugene OR) Wells K. Sam (Veneta OR) Haugland Richard P. (Eugene OR)
출원인 / 주소
Molecular Probes, Inc. (Eugene OR 02)
인용정보
피인용 횟수 :
50인용 특허 :
0
초록▼
The invention relates to a method of analyzing a sample thought to contain bacteria using an aqueous solution comprising one or more fluorescent dyes: a fluorescent dye of formula I, a fluorescent dye of formula II, a fluorescent dye of formula III, and a fluorescent dye of formula IV. Each of the d
The invention relates to a method of analyzing a sample thought to contain bacteria using an aqueous solution comprising one or more fluorescent dyes: a fluorescent dye of formula I, a fluorescent dye of formula II, a fluorescent dye of formula III, and a fluorescent dye of formula IV. Each of the dyes differ each from the other in their affinity for nucleic acids and in their spectral response to different types of bacteria in the sample. The first three dyes are nucleic acid stains and the fourth dye is a fluorescent reagent that binds selectively to cell surface components. The fluorescent dyes of formula I are highly membrane-permeant cyanine dye derivatives and label all bacteria, whether live or dead, whether Gram positive or Gram negative. The dyes of formula II label only live Gram positive bacteria and label all dead bacteria, whether Gram positive or negative. The dyes of formula II bind to nucleic acids preferentially with respect to the dyes of formula I. Fluorescent formula III dyes are membrane impermeant dyes that give a fluorescent signal only in cells with compromised plasma membrane integrity, whether Gram negative or Gram positive, and have a much higher binding affinity for nucleic acids than the fluorescent dyes of either formula I or formula II. Formula IV fluorescent dyes preferentially bind to an exterior component of a bacterium. The dyes are combined with a sample suspected of containing bacteria and illuminated at an appropriate wavelength to differentiate, according to the fluorescence response, live Gram negative, dead Gram negative, live Gram positive and dead Gram positive bacteria in the sample.
대표청구항▼
A method of analyzing bacteria for Gram reaction, comprising: a) combining a bacteria sample, simultaneously or sequentially, with an aqueous dye solution comprising a fluorescent dye of formula I, in combination with an aqueous dye solution comprising a fluorescent dye of formula II or an aqueous d
A method of analyzing bacteria for Gram reaction, comprising: a) combining a bacteria sample, simultaneously or sequentially, with an aqueous dye solution comprising a fluorescent dye of formula I, in combination with an aqueous dye solution comprising a fluorescent dye of formula II or an aqueous dye solution comprising a fluorescent dye IV or both, where each dye is present in solution in an amount sufficient to give a detectable fluorescent response and where each dye present in solution is selected such that its detectable fluorescent response, after staining and illumination of the sample, is different from the fluorescent response of any other dye being combined; wherein said dye of formula I has the formula [Figure] wherein each R1 is independently H; or an alkyl group having from 1-6 carbons; or a trifluoromethyl; or a halogen; or －OR8, －SR8 or －(NR8R9) where R8 and R9, which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic, heteroalicyclic, aromatic or heteroaromatic rings, containing 1-4 heteroatoms, wherein the hetero atoms are O, N or S: or R8 and R9 taken in combination are －(CH2)2－L－(CH2)2－where L=a single bond, －O－, －CH2－, or －NR10－where R10 is H or an alkyl group having 1-6 carbons; and t=1-4; R2 is an alkyl group having 1-6 carbons; X is O, S, Se or NR15, where R15 is H or an alkyl group having 1-6 carbons; or X is CR16R17 where R16 and R17, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R16 and R17 taken in combination complete a five or six membered saturated ring:. n=0, 1 or 2; Z-is a biologically compatible counterion; O has the formula Q1 or Q2 [Figure] (Q1) [Figure] (Q2) wherein Y is －CR3〓CR4－; p and m=0 or 1, such that p+m=1; R5 is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or R5 is an OMEGA; R3, R4, R6 and R7, which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or －OH, －OR8, －SR8, －(NR8R9); or －OSO2R19 where R19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; or an OMEGA; or R6 and R7, taken in combination are －(CH2)v－where v=3 or 4, or R6 and R7 form a fused aromatic ring according to formula Q2; R11, R12, R13, and R14, which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or an OMEGA; or －OH, －OR8, －SR8, or －(NR8R9); OMEGA is a saturated or unsaturated, substituted or unsubstituted, cyclic substituent that has a total of 2-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms wherein the heteroatoms are O, N or S, that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, alkoxy or carboxyalkyl, having 1-6 carbons, and that is attached as R3, R4, R5, R6, R7, R11, R12, R13, or R14 by a single O bond; such that at least one of R3, R4, R5, R6, R7, R11, R12, R13, and R14 is an OMEGA, and, where more than one of R3, R4, R5, R6, R7, R11, R12, R13, and R14 is an OMEGA, each OMEGA is optionally the same or different; said dye of formula II has the formula [Figure] wherein Rii is a C4-C8 alkyl; and Z-is a biologically compatible counterion; and said dye IV is a fluorescent reagent that preferentially binds to an exterior component of Gram-positive bacterium; b) allowing sufficient time for each dye in solution to combine with one or more bacterium in the sample to give a dyed bacteria mixture; c) illuminating the dyed bacteria mixture at a suitable absorption wavelength that results in one or more illuminated bacterium; d) observing the illuminated bacterium with means for detecting the fluorescent response resulting from illumination; and e) analyzing the fluorescent response of said illuminated bacterium, wherein the fluorescent dye of formula I stains intracellular nucleic acids for all bacteria in the sample; the fluorescent dye of formula II stains intracellular nucleic acids for all Gram positive bacteria in the sample; and the fluorescent dye IV stains the external surface of all Gram positive bacteria in the sample.
연구과제 타임라인
LOADING...
LOADING...
LOADING...
LOADING...
LOADING...
이 특허를 인용한 특허 (50)
Kawashima, Yasuyuki, Apparatus and method for analyzing bacteria.
Williams, Gregory B.; Nothaft, Daniel M.; Enscoe, Glenn F.; Burtner, Kathleen N.; Kangas, Monte E., Combined rapid susceptibility assay and microorganism identification system.
Bradford, Jolene A.; Cheung, Ching Ying; Huang, Shih Jung; Pinson, Patrick R.; Yue, Stephen T., Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use.
Bradford, Jolene; Cheung, Ching-Ying; Huang, Shih-Jung; Pinson, Patrick; Yue, Stephen, Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use.
Bradford, Jolene; Cheung, Ching-Ying; Huang, Shih-Jung; Pinson, Patrick; Yue, Stephen, Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use.
Bradford, Jolene; Cheung, Ching-Ying; Huang, Shih-Jung; Pinson, Patrick; Yue, Stephen, Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use.
Krahn, Thoams; Paffhausen, Wolfgang; Schade, Andreas; Bechem, Martin; Schmidt, Delf, Masking of the background fluorescence and luminescence in the optical analysis of biomedical assays.
Wainwright, Norman R.; Manian, Bala S.; Stimpson, Eric; Kolonia, Brian J.; Kolonia, Robert K., Optical method for detecting viable microorganisms in a cell sample.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.