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AssigneeThe United States of America as represented by the Secretary of the Navy
InventorsFrances S. Ligler, James R. Campbell
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Biological toxins are indirectly detected by using polymerase chain reaction to amplify unique nucleic acid sequences coding for the toxins or enzymes unique to toxin synthesis. Buffer, primers coding for the unique nucleic acid sequences and an amplifying enzyme are added to a sample suspected of containing the toxin. The mixture is then cycled thermally to exponentially amplify any of these unique nucleic acid sequences present in the sample. The amplified sequences can be detected by various means, including fluorescence. Detection of the amplified sequences is indicative of the presence of toxin in the original sample. By using more than one set of labeled primers, the method can be used to simultaneously detect several toxins in a sample.
A method for detecting a DNA coding for a toxin of biological origin or a DNA coding for an enzyme specific to toxin production, said toxin suspected of being present in a sample known not to contain any organisms which produce said toxin, comprising the steps of:
A method for detecting a plurality of DNAs coding for toxins of biological origin or DNAs coding for enzymes specific to production of said toxins in a sample suspected of including at least one of said toxins and known not to contain any organisms which produce said at least one toxin, comprising the steps of:
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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
It is known that large quantities of proteins and small molecules can be produced by fermentation. For pharmaceutical applications, the products are carefully purified and screened to remove residual DNA. On the other hand, if the products are to be used for environmental applications, such as bioremediation or biological warfare, large quantities are made but rigorous purification is not performed, because costs are too high and there is no need for purification. The DNA coding for the product or for proteins (enzymes) that make the product remains with the product as a contaminant. The DNA is usually at too low a concentration to be detected by conventional methods.
Methods are known for multiplying the DNA, such as the recombinant and cloning techniques discussed in U.S. Pat. Nos. 4,675,283, Roninson; 4,925,792, Rappuoli; or 4,666,837 and Harford et al. However, such methods are not very fast, and the amount of the DNA produced still may not be enough to ensure accurate detection of the genetic material.
Another available method of amplification is known as polymerase chain reaction (“PCR”). With PCR nucleic acid sequences can be quickly and exponentially amplified for facilitated detection of the sequences.