Methods and articles for detecting deoxyribonuclease activity

A deoxyribonucleic acid and enzyme activity technology, applied in the field of detection of deoxyribonuclease activity and products, can solve the problems of laborious cultivation methods and other problems

Inactive Publication Date: 2012-01-25
3M INNOVATIVE PROPERTIES CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Culture methods often require laborious procedures to prepare agar which, once prepared, must be used within a relatively short period of time

Method used

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  • Methods and articles for detecting deoxyribonuclease activity
  • Methods and articles for detecting deoxyribonuclease activity
  • Methods and articles for detecting deoxyribonuclease activity

Examples

Experimental program
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example 1

[0144] Detection of Growth and DNase Activity in PETRIFILM Aerobic Counting Plates

[0145] Phosphate buffered saline (PBS) was obtained as a 1OX concentrate (OmniPur 6505; EMD Chemicals; Gibbstown, NJ). DNA (sodium salt, from salmon testes) and methyl green dye (zinc chloride salt, Aldrich 198080-10G) were obtained from Sigma-Aldrich (St. Louis, MO). Lambda-carrageenan (viscarin GP type 109F) was obtained from FMC BioPolymer (Philadelphia, PA). 3M PETRIFILM Aerobic Counting Plates and PETRIFILM Plate Reader (PPR) were obtained from 3M Company (St. Paul, MN). Staphylococcus aureus ATCC 25923 (DNase positive, methicillin sensitive) was obtained from the American Type Culture Collection (Manassas, VA). S. epidermidis strain 472 (DNase negative) and S. aureus strain 565 (DNase positive, methicillin resistant) were obtained from clinical isolates.

[0146] A solution of DNA-PBS solution was prepared by adding 2.0 mg / mL DNA and 0.1 mg / mL lambda carrageenan to IX (10 mM) PBS. ...

example 2

[0150] Analysis of Petrifilm plate images

[0151] PETRIFILM Aerobic Count Plates were prepared as described in Example 1. Plates were inoculated with S. aureus Staphylococcus ATCC 25923 (a DNase producing microorganism). After seeding, the hydrogel in the plate contained 20 μg / mL methyl green. Plates were imaged using a PETRIFILM plate reader as described in Example 1.

[0152] Red, green and blue images were exported to IMAGE-PRO Plus version 6.3.0.512 software. Illustrations of grayscale images of the board when illuminated with green or red LEDs are in Figure 7a and 8a displayed in . The graticules of the plate (which are clearly visible in the green illuminated image but not in the red illuminated image) have been omitted from the figure.

[0153] Figure 7a The hydrogel 782 in the inoculated portion of the plate is shown. Several bacterial colonies 784 are also shown. Using the line profile tool of Image Pro Plus software, the region of interest from pixel x,...

example 3

[0160] Early Detection of DNase-producing Microorganisms

[0161] PBS-DNA solutions with (50 μg / mL) and without methyl green were prepared as described in Example 1. Use a final methyl green concentration of 20 µg / mL in this experiment in all plates containing methyl green.

[0162] A suspension of washed bacterial cells (S. aureus ATCC strain 25923) was prepared as described in Example 1. The suspension was diluted and inoculated into Petrifilm aerobic count plates as described in Example 1 .

[0163] The inoculated plate was placed on top of a metal heat block set at 37°C. A NIKON E8400 digital camera (Nikon, Inc., Melvelle, NY) was placed on top of the plate. The camera was connected to a computer via IMAGE-PRO Plus version 6.3.0.512 software, which was programmed to take digital photographs of each plate every 30 minutes for 24 consecutive hours. Place an insulated lid over the camera and heat block apparatus to maintain the temperature of the plate at 37 °C. The pr...

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Abstract

The disclosure provides articles and methods useful for detecting a discrete source of DNase activity. DNase-producing microorganisms can be detected. The device can further include selective agents and/or indicators to differentiate groups or species microorganisms. Methods of use include detecting or enumerating DNase- producing microorganisms.

Description

[0001] Cross references to related patent applications [0002] This application claims the benefit of US Provisional Patent Application No. 61 / 155,666, filed February 26, 2009, which is hereby incorporated by reference. Background technique [0003] DNA nucleases (DNases) are ubiquitous in biological cells and are involved in many cellular functions including, for example, DNA replication and repair. Certain microorganisms (eg, Branhamella catarrhalis, Staphylococcus aureus, Serratia marcescens, Streptococcus pyogenes, Bacillus subtilis ), Vibrio alginolyticus, Cryptococcus neoformans, and Fusarium solani can produce DNase that is secreted out of cells. [0004] DNA nucleases (DNases) are used in many research and diagnostic applications. A DNase activity assay has been developed to monitor this enzyme. In research applications, DNase assays often employ radiolabeled DNA substrates and require separation of reacted products from unreacted substrate before enzymatic activi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12Q1/14
CPCC12Q1/34C12Q1/04G01N2333/922G01N2333/31C12N11/04
Inventor 帕特里克·A·玛奇米歇勒·L·罗索尔迈克尔·E·休斯
Owner 3M INNOVATIVE PROPERTIES CO
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