EP0285439A2
Expired / Lapsed
EP
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Techniques for preparing specimens for bacterial assays
Assignee: Gen Probe Inc
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Filing: 1988-03-31
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Granted: —
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Expires: 2008-03-31
Abstract
Abstract Methods and compositions are disclosed for improved liquification of mucoid secretion specimens by treatment with disulfide bond reducing agents and/or DNA digestion agents, and for improved concentration of selected bacterial species from such specimens, by concentrating white blood cells in the specimen associated with the bacterial species. The procedures have applicability to bacterial assay techniques including nucleic acid hybridization, culture and stain techniques.
Inventors
- 👤 Daniel Louis Kacian
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-24 17:45
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6888; C12Q1/689; C12Q1/02; C12Q1/24; C12Q1/6806; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/5002; G01N33/5005; G01N33/5094; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S435/8215; Y10S435/822; Y10S435/863; Y10S435/961; Y10S435/962; Y10S435/975
Claims Analysis
Total Claims: 38
Claim 1
Independent Claim
61 words
A method of liquifying a mucoid secretion specimen or other viscous biological specimen, comprising the steps of: (a) contacting said specimen with a disulfide bond reducing agent so as to cleave disulfide bonds in proteinaceous molecules in said specimen; and (b) contacting said specimen with a DNA digestion agent so as to cleave phosphodiester bonds in DNA molecules in said specimen.
Claim 2
Independent Claim
61 words
A method of liquifying a mucoid secretion specimen or other viscous biological specimen, comprising the steps of: (a) contacting said specimen with a disulfide bond reducing agent so as to cleave disulfide bonds in proteinaceous molecules in said specimen; and (b) contacting said specimen with a DNA digestion agent so as to cleave phosphodiester bonds in DNA molecules in said specimen.
Claim 3
Dependent Claim
13 words
The method of claim 1 wherein said disulfide bond reducing agent is dithiothreitol.
Claim 4
Dependent Claim
15 words
The method of claim 1 or 2 wherein said DNA digestion agent is deoxyribonuclease I.
Claim 5
Independent Claim
18 words
The method of any one of the preceding claims wherein said mucoid secretion specimen is a sputum specimen.
Claim 6
Dependent Claim
56 words
In a kit for assaying a mucoid secretion or other viscous biological specimen for the presence of a selected biological species, which kit includes a means for liquifying said specimen prior to assaying for said species, the improvement comprising a means in said kit for liquifying said specimen according to any one of the preceding claims.
Claim 7
Dependent Claim
15 words
The improvement of claim 5 wherein the selected biological species is a selected bacterial species.
Claim 8
Dependent Claim
18 words
The improvement of claim 6 wherein said selected bacterial species is a mycobacterium or an enteric bile-resistant pathogen.
Claim 9
Dependent Claim
78 words
In a method for assaying a mucoid secretion specimen, or other viscous biological specimen, for the presence of a selected bacterial species, which method comprises the steps of liquifying said specimen prior to assaying for said species and assaying for said species using a nucleic acid hybridization technique, the improvement comprising liquifying said specimen by contacting said specimen with a digestion mixture comprising a DNA digestion agent capable of cleaving phosphodiester bonds in DNA molecules in said specimen.
Claim 10
Dependent Claim
78 words
In a method for assaying a mucoid secretion specimen, or other viscous biological specimen, for the presence of a selected bacterial species, which method comprises the steps of liquifying said specimen prior to assaying for said species and assaying for said species using a nucleic acid hybridization technique, the improvement comprising liquifying said specimen by contacting said specimen with a digestion mixture comprising a DNA digestion agent capable of cleaving phosphodiester bonds in DNA molecules in said specimen.
Claim 11
Dependent Claim
13 words
The method of claim 8 wherein said DNA digestion agent is deoxyribonuclease I.
Claim 12
Dependent Claim
26 words
The improvement of claim 8 or 9 wherein said digestion mixture further comprises a disulfide bond reducing agent capable of cleaving disulfide bonds in said specimen.
Claim 13
Dependent Claim
13 words
The method of claim 10 wherein said disulfide bond reducing agent is dithiothreitol.
Claim 14
Dependent Claim
81 words
In a method for assaying a biological specimen for the presence of a selected bacterial species, which method includes the steps of concentrating the selected bacterial species from said biological specimen into a concentrated specimen and assaying said concentrated specimen for the presence of said selected bacterial species, the improvement comprising the step of forming said concentrated specimen by concentrating white blood cells from said biological specimen, which white blood cells are associated with a portion of said selected bacterial species.
Claim 15
Dependent Claim
81 words
In a method for assaying a biological specimen for the presence of a selected bacterial species, which method includes the steps of concentrating the selected bacterial species from said biological specimen into a concentrated specimen and assaying said concentrated specimen for the presence of said selected bacterial species, the improvement comprising the step of forming said concentrated specimen by concentrating white blood cells from said biological specimen, which white blood cells are associated with a portion of said selected bacterial species.
Claim 16
Dependent Claim
14 words
The improvement of claim 12 wherein said biological specimen comprises a mucosal secretion specimen.
Claim 17
Dependent Claim
12 words
The improvement of claim 13 wherein said mucosal secretion specimen comprises sputum.
Claim 18
Dependent Claim
23 words
The improvement of any one of claims 12 to 14 wherein said selected bacterial species is a mycobacterium, or an enteric bile-resistant pathogen.
Claim 19
Dependent Claim
16 words
The improvement of any one of claims 12 to 15 wherein said concentrating step comprises centrifugation.
Claim 20
Dependent Claim
21 words
The improvement of any one of claims 12 to 16 wherein said concentrated specimen comprises substantially cellularly intact white blood cells.
Claim 21
Independent Claim
77 words
A method for concentrating a selected bacterial species from a biological specimen comprising the steps of (a) concentrating white blood cells, which white blood cells are associated with said selected bacterial species, from said biological specimen to form a concentrated specimen; and (b) lysing said white blood cells in said concentrated specimen with a lysing reagent to form a lysed concentrated specimen comprising lysed white blood cells and substantially cellularly intact bacteria of said selected bacterial species.
Claim 22
Independent Claim
77 words
A method for concentrating a selected bacterial species from a biological specimen comprising the steps of (a) concentrating white blood cells, which white blood cells are associated with said selected bacterial species, from said biological specimen to form a concentrated specimen; and (b) lysing said white blood cells in said concentrated specimen with a lysing reagent to form a lysed concentrated specimen comprising lysed white blood cells and substantially cellularly intact bacteria of said selected bacterial species.
Claim 23
Dependent Claim
14 words
The method of claim 18 wherein said biological specimen comprises a mucoid secretion specimen.
Claim 24
Dependent Claim
20 words
The method of claim 18 or 19 wherein said selected bacterial species is a mycobacterium, or an enteric bile-resistant pathogen.
Claim 25
Independent Claim
19 words
The method of any one of claims 18 to 20 wherein said lysing reagent comprises a salt of deoxycholate.
Claim 26
Independent Claim
25 words
The method of any one of claims 18 to 21 comprising the further step of liquifying said lysed concentrated specimen using a DNA digestion agent.
Claim 27
Dependent Claim
13 words
The method of claim 22 wherein said DNA digestion agent is deoxyribonuclease I.
Claim 28
Dependent Claim
24 words
The method of claim 22 or 23 wherein said lysing reagent is chosen from lysing reagents that do not inactivate said DNA digestion agent.
Claim 29
Independent Claim
19 words
The method of any one of claims 18 to 24 wherein said lysing reagent comprises a salt of deoxycholate.
Claim 30
Independent Claim
30 words
The method of any one of claims 18 to 25 wherein said liquifying step forms a liquified lysed concentrated specimen comprising substantially cellularly intact bacteria of said selected bacterial species.
Claim 31
Dependent Claim
84 words
In a kit for assaying a biological specimen for the presence of a selected bacterial species, which kit includes a means for concentrating the selected bacterial species from said biological specimen into a concentrated specimen, and optionally includes a means for liquifying said lysed concentrated specimen, the improvement comprising a means in said kit for forming said concentrated specimen and optionally a means in said kit for liquifying said lysed concentrated specimen according to the improvement of any one of claims 12 to 26.
Claim 32
Dependent Claim
66 words
A method for assaying mucoid secretion specimen or other biological specimen for the presence of a selected bacterial species comprising the steps of (a) concentrating the selected bacterial species from said mucoid secretion specimen or other biological specimen according to the method of claim 23 to form a lysed concentrated specimen; and (b) assaying said lysed concentrated specimen for the presence of said selected bacterial species.
Claim 33
Dependent Claim
22 words
The method of claim 28 wherein said assaying step comprises a nucleic acid hybridization step adapted to detect said selected bacterial species.
Claim 34
Dependent Claim
20 words
The method of claim 28 wherein said assaying step comprises a culturing step adapted to detect said selected bacterial species.
Claim 35
Dependent Claim
20 words
The method of claim 28 wherein said assaying step comprises a staining step adapted to detect said selected bacterial population.
Claim 36
Independent Claim
18 words
The method of any one of claims 28 to 31 wherein said specimen is a mucoid secretion specimen.
Claim 37
Independent Claim
23 words
The method of any one of claims 28 to 32 wherein said selected bacterial species is a mycobacterium or an enteric bile-resistant pathogen.
Claim 38
Independent Claim
17 words
The method of any one of claims 28 to 33 wherein said mucoid secretion specimen comprises sputum.
References Cited (Backward Citations)
No citation records indexed.
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