Botulism Strain Detection
Real Time Polymerase Chain Reaction Quadraplexed for Rapid Detection of Clostridium botulinum Toxin Types A, B, E, and F
The toxin produced by Clostridium botulinum (botulism) is one of the most potent toxins known. It is believed that only one gram, properly aerosolized and distributed, could kill an estimate 1.5 million people. Linked to food poisoning and sudden infant death syndrome (SIDS), this toxin, combined with its many means of contraction (ingestion, inhalation or wound infection) makes it an ideal biowarfare agent. The current method for detecting botulism strains is not fast enough to mount an effective public health response, were this toxin used in a bioterrorist attack.
Of the seven known forms, four of these (types A, B, E, and F) are known to cause human disease. This novel assay is a rapid, highly sensitive, highly reliable and robust method for detecting all four toxin types within a single RTPCR tube. Isolated DNA is combined with PCR primers and fluorescing probes both highly specific to each toxin type. The Smartcycler II both amplifies and detects the extent of florescence on the DNA.
Whether the reaction was run on one, two, three, or all four strains at once, each strain was detected, indicating that the primers and probes are specific enough to not interfere with each other. Real world epidemiological utility of this assay was measured by experimenting on isolated DNA from processed vegetable material spiked with C. botulinum bacteria. No false positives or false negatives were obtained at any point during experimentation.
This assay would be useful to any public health surveillance laboratory, such as the Centers for Disease Control. However, since this assay measures the DNA that codes for the C. botulinum toxin and not the toxin itself, and in highly purified biowarfare agents the toxin may be purified to undetectable amounts, it is suggested that this method be utilized in conjunction with other assays that test for protein toxins. These later methods are commercially available and currently being refined. In an anticipated outbreak situation however, having to double test due to refined DNA would not be a concern.
David O. Pickett, Richard A. Robison
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