Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-09-01
2002-05-28
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S034000, C435S039000, C435S004000, C435S018000, C435S023000, C435S287100, C435S287300, C435S287400, C435S288700, C435S008000, C422S051000
Reexamination Certificate
active
06395504
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for the use of a phage associated lysing enzyme for the detecting the presence and determining the quantity of bacteria present in or on a wide variety of substances.
BACKGROUND OF THE INVENTION
Microbial contamination of surfaces and substances is a significant cause of morbidity and mortality. Rapid and routine procedures for quantitative determination of bacteria present on surfaces is frequently of vital importance, particularly in food processing, drinking water, and in hospitals. Food poisoning is often a result of microbial contamination of meat or food that occurs during processing. Contamination can be spread through contact of food with surfaces. In addition, spread of disease in hospitals and other facilities often occurs as a result of passage of infectious microbes on the surface of clothes or equipment.
A key feature of these applications is the requirement for rapid testing within minutes, a method that will overcome the potential contaminants from a variety of surfaces, a requirement for no cross-over in the results from one test to a second, and a need for both general and specific testing for microbes, that is, the ability to test for contamination by both microbial counts and the ability to test for the presence of specific microbes.
Various methods have been utilized to measure microbial contamination on surfaces. Traditional procedures for assaying bacteria on surfaces are based on swabbing the surface followed by a culture of the swab for 24 to 48 hours in or on media that supports the growth of microbial species. The cultures are observed manually or with automated instrumentation to determine the number of colonies that have formed as an indicator of the number of microbes initially present on the surface. The disadvantages of this methodology are long assay times, requirements for specially trained personnel, and possible inadequate identification of the presence of certain potentially pathogenic microbes whose growth is not supported by the specific media or environment. In particular, it may be difficult to detect fungal contamination by this method. In addition, in many of the potential applications, the method does not give results in the time frame required for effective response.
Luminescent reactions have been utilized in various forms to detect bacteria in fluids and in processed materials. In particular, bioluminescent reactions based on the reaction of adenosine triphosphate (ATP) with luciferin in the presence of the enzyme luciferase to produce light (the “firefly” reaction) have been utilized. Since ATP is present in all living cells including all microbial cells, this method can be used in a rapid assay to obtain a quantitative estimate of the number of living cells in a sample. Early discourses on the nature of the reaction, the history of its discovery, and its general area of applicability are provided by E. N. Harvey (1957), A History of Luminescence: From the Earliest Times Until 1900, Amer. Phil. Soc., Philadelphia Pa and W. D. McElroy and B. L. Strehler (1949) Arch. Biochem. Biophys. 22:420-433. Alternatively, chemiluminescent detection by isoluminol or similar compounds has been used. This method is based on the detection of iron-containing substances in microbes.
Test procedures exemplifying the use of bioluminescent reactions for bacterial determinations and, specialized instrumentation for measurement of the associated light emission, are known and have been disclosed. Plakas (U.S. Pat. Nos. 4,013,418, 4,144,134, and 4,283,490) teaches a bioluminescent assay for the detection of bacteria in a sample including the steps of lysing non-bacterial cells, effecting filtration by positive pressure, washing, lysing bacterial cells and detecting ATP released with a luciferin/luciferase/Mg2+ regent. The art in this patent does not deal with the specific problems associated with collection of material from a surface or with the detection of specific bacteria. No issue of the timing is mentioned and the invention as disclosed would require significant time.
Chappelle in U.S. Pat. No. 4,385,112 discloses a method for detection of bacteria in water based on bioluminescence. This test requires several hours to perform and is specifically addressed to the detection of total bacterial content in water.
Miller (PCT application U.S. Ser. No. 88/00852) discusses a similar assay for use with urine samples, but does not discuss the issues of collection from a surface and the assay timing is not specifically set forward in this application. further, no method for detection of specific bacteria is elucidated.
U.S. Pat. No. 3,933,592 (Clendenning) discusses a method for bioluminescent detection of microbial contamination and in the examples refers to performing the procedure in less than 2 minutes. The procedure does not involve pre-treatment phases and the removal of somatic cell ATP.
U.S. Pat. No. 5,258,285 (Aegates) discloses a method for detection of bacterial concentration in a sample that utilizes a filtration step, a washing step to remove extraneous material including somatic cell ATP, establishing an extraction chamber in which luciferin/luciferase/Mg2+ is added and the reaction measured. This method does not mention time. In addition, it utilizes separate chambers for washing, extracting the bacterial ATP, and measuring the reaction. This may potentially result in decreased sensitivity due to loss of the material in the process of transferring the solution from chamber to chamber. Further, the method does not describe a means of collecting a sample from a surface.
U.S. Pat. No. 5,736,351 (Miller et al) discloses a method and device for determining the presence and concentration of total microbial contamination or the presence and concentration of a specific microbial species on a surface is described. The method consists of a means of a collection device and fluid for removing the microbes from the surface and suspending them in a fluid phase. An aliquot of the fluid phase is introduced into a disposable test ticket which allows filtration of the sample to remove extraneous substances including somatic cells, and concentration of the microbes. The total concentration of microbes is determined by adding a somatic and bacterial releasing reagent to a disposable test device which comprises a membrane containing the luminescent reagents luciferin and luciferase, and introducing the disposable test device into a luminometer that can read the luminescence from the underside.
SUMMARY OF THE INVENTION
The present invention is a method and device for detecting the presence and/or concentration of specific bacteria found on surfaces, in or on food, in the air, water, or in biological samples, by the use of bioluminescence. This device and method relies on the use of at least one lytic enzyme produced by a bacteria infected with a bacteriophage specific for the bacteria to lyse the bacteria, allowing for the release of ATP in the bacteria and its detection by chemical-bioluminescence analysis. Unlike the method and device of U.S. Pat. No. 5,5736,351, the lytic enzyme can be used in place of the bacterial releasing agent, and it may not even be necessary to use the somatic releasing agent, thereby simplifying the procedures.
Reports have described the characteristics of an enzyme produced by the group C streptococcal organism after being infected with a particular bacteriophage identified as C
1
(Maxted, W. R. “The Active Agent in Nascent Page Lysis of Streptococci,” J. Gen Micro., vol 16, pp 585-595, 1957, Krause, R. M., “Studies on the Bacteriophages of Hemolytic Streptococci,” J. Exp. Med, vol. 108, pp 803-821, 1958) and Fischetti, (Fischetti, V. A., et al, “Purification and Physical Properties of Group C Streptococcal Phage Associated Lysin,” J. Exp. Med, Vol 133 pp. 1105-1117, 1971). The enzyme was given the name lysin and was found to specifically cleave the cell wall of group A, group C, and group E streptococci. These investigators provided information on the characteris
Grant Jonathan
Grant Patent Services
Leary Louise N.
New Horizons Diagnostics Corp.
LandOfFree
Use of phage associated lytic enzymes for the rapid... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Use of phage associated lytic enzymes for the rapid..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Use of phage associated lytic enzymes for the rapid... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2852502