Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-04-21
2001-11-06
Horlick, Kenneth R. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S252100, C536S024320, C536S024330
Reexamination Certificate
active
06312891
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to species-specific oligonucleotide probes for binding specifically to ribosomal RNA of bacterium
Vibrio Vulnificus
or
Vibrio cholerae
. The present invention further relates to methods and kits for identifying the bacteria
Vibrio vulnificus
or
Vibrio cholerae
with such probes in a single day, without the need for culturing the bacteria.
BACKGROUND OF THE INVENTION
Vibrio vulnificus
and
Vibrio cholerae
are small organisms called bacteria that live in the marine environment.
Vibrio cholerae
can also survive in fresh water. By drinking water, eating fruits and vegetables, fish or shellfish that are contaminated with this bacterium (one bacteria), a person can become very ill or may even die from the disease cholera which causes severe diarrhea and dehydration.
Vibrio vulnificus
can cause serious illness and even death within three days in people who eat raw or improperly cooked fish or shellfish that are infected with this microorganism.
There are many types of bacteria, both good and bad, in food and water. To find out whether water or food contains these harmful Vibrio bacteria, or if a person is infected with them, laboratory tests must be performed. The first step is to culture, or grow, the bacteria in a special liquid. Then a series of tests are done to identify the bacteria based on whether or not they use certain sugars and other compounds in order to grow. It may take as long as one week to do these tests and by that time a person may die if not given the proper medicine. What is needed is a rapid and easy way to detect and identify
Vibrio cholerae
and
Vibrio vulnificus.
A faster way to check for the presence of these bacteria is to use a molecule called an antibody that recognizes then binds to a specific part of the bacterium. See, for example, Tamplin, M. L. et al.:
Applied and Environmental Microbiology
, 57:1235-1240 (April 1991), which reports an enzyme immunoassay for the identification of
Vibrio vulnificus
in seawater, sediment and oysters. However, this method also requires growing the bacteria first and still takes several days to do. Also, antibodies may be too specific and detect only those bacteria from one source but not from another.
Another method involves the use of a “gene probe” to identify the bacteria. Every living organism has molecules of DNA (deoxyribonucleic acid) which contains instructions for each cell to make the things it needs to sustain life. A gene is a piece of DNA that instructs the cell to make one particular type of protein molecule. A gene probe is also made of DNA. See, for example, Amann, R. et al.:
Nature
. 351:161-164 (May 9, 1991); and Amann, R. et al.:
J. Bacteriology
. 173:762-770 (February 1990).
Vibrio vulnificus
and
Vibrio cholerae
have specific genes that make proteins which are responsible for causing sickness. A gene probe that specifically seeks out the genes for these toxic proteins can be used to find out whether these bacteria are in food, water or people. However, there is a problem with using a gene probe. Bacteria are one-celled organisms. Each bacterium has only one gene for each kind of protein it makes. It is difficult to detect the one and only toxic protein gene in each cell.
One way to solve this problem is to use a method called PCR to amplify (make many copies of) the gene in a test tube and then use the gene probe to find the gene copies. This method, however, is sophisticated and time-consuming and requires a sufficient number of bacteria to perform the method.
With the current outbreak of Vibrio cholerae in South America, it is apparent that a rapid means of detection is warranted. Consequently, there is a need in the industry for simple, but quick and accurate tests for the reliable and early detection of the bacteria
Vibrio vulnificus
or
Vibrio cholerae
in, for example, water, food, blood, feces, and the like, contaminated with such bateria.
SUMMARY OF THE INVENTION
In brief, the present invention alleviates and overcomes certain of the above-referenced problems and shortcomings of the present state of the art through the discovery of novel oligonucleotide probes which are species-specific for the 23S rRNA of
Vibrio vulnificus
and
Vibrio cholerae
. Generally speaking, the DNA probes of the present invention seek out and hybridize with specific, unique RNA regions of each 23S ribosome in every cell of either the bacteria
Vibrio vulnificus
or the bacteria
Vibrio cholerae
. The probes of the present invention are uniquely made of very short pieces of DNA, so small that they can easily enter a
Vibrio vulnificus
or
Vibrio cholerae
cell, respectively.
The DNA probes of the instant invention are amazingly versatile in that they can be mixed with for example, water, food such as fish or shellfish, blood, feces or other type of samples to be tested. To identify the bacteria, a labeled moiety, such as a dye, a biotin or a radioisotope molecule, is attached to one end of each DNA probe. When a fluorescent dye is selected and a light is shone on the sample (food, water, fish, shellfish, blood, feces, etc.), the fluorescent dye will glow only if the bacteria for which the DNA probe is specific are present. Quite amazingly, it is actually possible to see each tiny bacterium glowing by using an epifluorescent microscope. And, because there is no need to culture or grow the bacteria beforehand or to amplify their genes, the tests of the present invention are simpler, much quicker and more reliable than the other kinds of test which have been available heretofore for detecting
Vibrio vulnificus
and
Vibrio cholerae.
The methods of the present invention involve fixation of whole bacterial cells from a mixed culture sample on, for example, membrane filters or microscope slides followed by hybridization with tagged species-specific probe, and washing to remove excess or non-specifically bound probe. The presence or absence of
Vibrio vulnificus
or
Vibrio cholerae
cells is directly determined by, for example, epifluorescence microscopy when the DNA probes are labeled with a fluorescent dye.
Each probe of the instant invention is a single-stranded oligodeoxynucleotide sequence, approximately 15-20 bases in length, which specifically targets the ribosomal RNA (rRNA) component of the ribosomes within
Vibrio cholerae
or
Vibrio vulnificus
cells.
While the DNA probes of the present invention typically include between about 15 and about 20 nucleotides, it should nevertheless be understood by those skilled in this art that the present invention contemplates DNA probes of any size so long as the objectives of the present invention are not defeated, i.e., the DNA probes are of a size sufficient so that they can enter the bacteria cells, and the DNA probes include an effective number of nucleotides so that they can hybridize specifically with the ribosomal RNA of either the bacteria
Vibrio vulnificus
or the bacteria
Vibrio cholerae
, respectively.
Accordingly, it can now be appreciated that the present invention is believed to provide a valuable and worldwide solution to the
Vibrio vulnificus
and
Vibrio cholerae
art that has long sought effective and inexpensive means to quickly and reliably detect such pathogenic bacteria. As a result, the present invention can help to protect the public health, and perhaps prevent future epidemics, by its ability to detect infection in humans as well as in contaminated water and food at an early state.
The above features and advantages will be better understood with reference to the Detailed Description set out hereinbelow. It will also be understood that the biological materials of this invention are exemplary only and are not to be regarded as limitations of this invention.
REFERENCES:
patent: 4582789 (1986-04-01), Sheldon, III et al.
patent: 5258284 (1993-11-01), Morris, Jr. et al.
patent: 5582993 (1996-12-01), Stackebrandt et al.
patent: 5607835 (1997-03-01), Reeves et al.
patent: 5137600 (1993-06-01), None
patent: 5276996 (1993-10-01), None
patent: 8803957 (1988-06-01), None
Dorsc
Bennison Brenda W.
LaRock Paul A.
Reeves Robert H.
Akerman, Senterfitt & Eidson
Florida State University
Horlick Kenneth R.
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