Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Statutory Invention Registration
1992-01-09
2001-08-07
Carone, Michael J. (Department: 3641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S183000, C536S023100, C536S024330
Statutory Invention Registration
active
H0001985
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to detecting biological toxins and, more particularly, to a method for amplifying genes coding for the toxins and detecting the amplified genes, which detection is indicative of the presence of the toxins.
2. Description of the Related Art
The ability to detect the presence or absence of biological toxins, such as botulin (a toxin produced by the bacteria
Clostrium botulinum
) or aflatoxins (a toxin produced by the fungus
Aspergillus favus
) contaminating food, various biological warfare agents contaminating the air, and various contaminants of water or biological samples, has been a desire of scientists for ages. Such an ability would help prevent disease, incapacitation or other maladies attributable to the toxins, and would be useful in countering the use of biological weapons.
Current tests for detecting toxins usually depend on chromatographic techniques, such as gas or high-pressure-liquid chromatography, mass spectroscopy, in vivo tests or in vitro bioassays. In vivo tests involve injecting the toxin into animals, usually mice, at varying doses to determine lethality. In vitro bioassays rely on the use of an antibody, receptor or living cell that binds the toxin directly. Thus, the actual toxin is sought to be detected.
In other cases, assay methods are used to detect an organism which is the source of the toxin. For example, if a pathogenic organism is known to be associated directly with a toxin, tests have sought to detect the presence of the pathogen. Such tests may include culturing the live organism.
The conventional tests described above generally are not very sensitive. They also may require pre-concentration of a sample suspected of containing the toxin or organism. Further, in some cases, at least partial purification is necessary to remove interfering substances. These steps can be time-consuming and costly.
To date, there has not been developed a quick and efficient method for detecting the presence of a biological toxin in a sample, said detection being possible even if the toxin has been denatured or the organism responsible for producing the toxin is no longer present.
SUMMARY OF THE INVENTION
Accordingly, it is a purpose of the present invention to provide a more sensitive method for detecting biological toxins.
It is another purpose of the present invention to detect toxins in the food service, food preparation and dairy industries.
It is another purpose of the present invention to detect toxins used as offensive biological weapons.
It is another purpose of the present invention to detect genes that have been willfully engineered into otherwise innocuous microorganisms (“weaponized organisms”).
It is another purpose of the present invention to provide a method for detecting a biological toxin by testing for the presence of the unique nucleic acid sequence responsible for the synthesis of the toxin or a specific protein (enzyme) used to produce the toxin.
It is another purpose of the present invention to detect a plurality of different biological toxins in the same sample, simultaneously.
It is another purpose of the present invention to detect target nucleic acid sequences using a simple and easy to use method, which detection is indicative of the presence of toxins coded by the sequences.
It is another purpose of the present invention to solve the problems associated with conventional detection of low amounts of biological toxins, of the biological entity responsible for producing the toxin, such as a microorganism, or of the nucleic acids coding for these toxins.
It is another purpose of the present invention to detect the presence in environmental and biological samples, of specific genes that encode toxins, regardless of whether the gene is present in its normal genetic host, or whether it has been inserted into a different host by genetic engineering or by a random, natural gene transfer event.
It is another purpose of the present invention to provide a method for identifying toxins in environmental or biological samples by detecting minute traces of genetic material that encode said toxins, and which are found in association with said toxin preparations as a byproduct of the toxin manufacturing process, such as toxins produced for dissemination as offensive biological weapons.
To achieve the foregoing and other purposes of the present invention there is provided the following method for detecting biological toxins in samples. Instead of trying to directly detect the toxin present, the nucleic acid coding for the toxin is amplified using the known method polymerase chain reaction (“PCR”), and the amplified nucleic acid is then detected as an indication of the presence or absence of the toxin.
More particularly, samples, such as clinical, food, water supply or air samples, suspected of containing biological toxins are collected. With the unique gene sequence coding for the toxin being known, a primer pair can be selected which is complementary to the gene sequence. The primers and an appropriate enzyme for amplification are then mixed with the sample. The preparation is then placed into a thermal cycler, wherein PCR amplifies any toxin gene present. Thereafter, methods are used to detect the amplified gene. Detection of the gene coding for a toxin is indicative of the presence of the toxin in the original sample.
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Olive, D. M. Detection of EnterotoxigenicEscherichia coliafter Polymerase Chain Reaction Amplification . . . J. Clin. Microbiol. (Feb. 1989) 27:261-265.*
Locus CBBOTAG from EMBL data library (Created May 7, 1990).
Campbell James R.
Ligler Frances S.
Baker Aileen J.
Carone Michael J.
Edelberg Barry A.
Ressing Amy Loch
The United States of America as represented by the Secretary of
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