Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1992-12-07
2002-04-02
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S007100, C435S007200, C435S007300, C435S007370, C435S007920, C435S029000, C435S033000, C435S038000, C435S040000, C435S848000, C435S849000, C435S287100, C436S177000, C436S178000, C436S809000
Reexamination Certificate
active
06365368
ABSTRACT:
BACKGROUND OF THE INVENTION
Detection and enumeration of indicator bacteria is of primary importance for monitoring the sanitation and microbiological quality of food and water. The presence of the bacterium
Escherichia coli
the most common fecal indicator, has been used in the past 100 years to monitor incidences of sewage contamination.
Current detection methodologies for
E. coli
or fecal coliforms are based on the properties of acid or gas production from the fermentation of lactose. The most widely used methods are: the Most Probable Number (MPN) assay and the Membrane Filtration (MF)test. Both techniques are approved by the Environmental Protection Agency (EPA) and the American Public Health Association for the microbiological examination of water and waste water.
The MPN method is actually comprised of 3 separate assays. In the Presumptive test, a nonselective medium such as Lauryl Sulfate Tryptose broth (LST) or Lactose broth is used to check for gas production from the fermentation of lactose. Gas positive tubes are then subcultured into a more selective medium, Brillant Green Lactose Bile (BGLB) for coliforms or
E. coli
broth (EC) for fecal coliforms, and again checked for gas production (confirmed test). Samples from positive confirmatory tests are required to be tested further, by plating on a selective and differential medium like Eosin Methylene Blue agar or Endos agar, followed by Gram Stain and biochemical tests to firmly establish the presence of the indicator bacterium (completed test). The entire MPN assay requires up to five days to complete; therefore, it is not cost effective in terms of both time and materials.
The MF techniques for the biological examination of water were introduced in early 1950's. Unlike the MPN assay which was tedious and time consuming, MF analysis could be completed in 24 hours without the need for further confirmations, and the system allowed for the examinations of large volumes of water samples. The basic MP procedure is as follows: a volume of sample, usually 100 ml is filtered through a 0.45 micron pore-diameter filter, and then incubated on a sterile pad saturated with selective medium. The two media most often used are the m-Endo broth, selective for coli-forms at 35° C., and the mFC broth, selective for fecal coliforms at 45° C. Both of these media have been reported to underestimate the actual numbers of bacteria present, due either to the selectivity of the medium or the high temperature used for incubation (45° C.). Such incidences of false negatives have been especially prevalent when the organisms in the sample have been sublethaly injured by environmental factors and/or chemicals.
Recently, modifications have been proposed by the EPA to follow up the MF assay with a confirmatory step using LST broth followed by BGLB broth as in the MPN test. Such modifications even though they would reduce the incidences of false positive and false negative reactions, they would triple the MF assay time from 24 hours to 72 hours.
In 1982, Feng and Hartman (Applied and Environmental Microbiology, Vol. 43 No. 6, June 1982 pp. 1320-1329) introduced a fluorogenic assay for the detection of
E. coli
using the substrate 4-methylumbelliferone glucuronide (MUG).
E. coli
cells produce the enzyme beta-D-glucuronidase which could cleave the substrate, releasing the fluorogenic methyl umbelliferone radical into the Presumptive LST medium. The assay provided both the Presumptive (gas production) and the Confirmed data (fluorescence) for fecal coliforms in a single test within 24 hours. Although the MUG assay was rapid and simple, only 90% of the
E. coli
cells tested produced this enzyme, hence the test was not 100% reliable.
It is evident that currently, no suitable assays exist for the enumeration of coliforms or fecal coliforms. The development of a simple, rapid and reliable detection assay would not only decrease cost and time, but also greatly increase the efficiency of monitoring water sanitation.
The detection system we propose is to use monoclonal antibodies to supplement existing assays. By incorporating the principles of acid and/or gas production from lactose, fluorescence from MUG, along with an Enzyme Linked Immuno-Sorbent Assay (ELISA) using an
E. coli
specific monoclonal antibody all within one test, we can derive several distinct data from a single assay, and provide a fairly reliable detection system for
E. coli.
SUMMARY OF THE INVENTION
The present invention concerns a method for analyzing a liquid sample to determine the presence of a specific microbe, e.g.
E. coli.
The method comprises randomly distributing a known volume of the liquid sample onto a suitable membrane material as a defined number of equal volume aliquots. Each of the aliquots is then filtered under appropriate conditions through the membrane material to collect the microbe on the membrane.
The microbe collected on the membrane material from each aliquot is separately contacted with a defined volume of a suitable solution containing a predetermined amount of detectable reagent specific for the microbe e.g. an antibody specific to an antigenic site on the microbe under conditions permitting formation of complexes between the reagent and the microbe.
By determining the amount of complex formed from each aliquot one can determine the amount of microbe originally present in the liquid sample.
In a specific embodiment of the invention additional steps can be added after collecting the microbe on the membrane and before contacting the microbe with the detectable reagent.
These steps comprise contacting the membrane material with a non-selective medium permitting growth of the microbe and incubating the membrane material in contact with the non-selective medium under conditions such that the microbe multiplies a predetermined number of times.
The membrane material in contact with the non-selective medium is then tested for one or more substances indicative of the presence of the microbe. After testing the non-selective medium is removed from contact with the membrane material.
The invention also concerns a device useful for analyzing a liquid sample to quantitatively determine the presence of a specific microbe in the liquid sample.
REFERENCES:
patent: 4443549 (1984-04-01), Sadowski
patent: 4493815 (1985-01-01), Fernwood et al.
patent: 4592994 (1986-06-01), Mattiason
patent: 4652533 (1987-03-01), Jolley
patent: 4704255 (1987-11-01), Jolley
Singer, R.H., et al.,Biotechniques, vol. 4, No. 3, pp. 230-243, 1986.*
Feng, P.C.S., et al, App. Environ. Microbiol., vol. 43, No. 6, Jun. 1982, pp. 1320-1329.
Feng Peter
Lobel Steven A.
Massey Richard
Minnich Scott A.
Schochetman Gerald
Chin Christopher L.
Evans, Esq. Barry
IGEN International Inc.
Kramer Levin Naftalis & Frankel LLP
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