AMP is released by means of heat shock, then detected via bioluminescence.
A method of rapid detection of bacterial spores is based on the discovery that a heat shock consisting of exposure to a temperature of 100 °C for 10 minutes causes the complete release of adenosine monophosphate (AMP) from the spores. This method could be an alternative to the method described in the immediately preceding article. Unlike that method and related prior methods, the present method does not involve germination and cultivation; this feature is an important advantage because in cases in which the spores are those of pathogens, delays involved in germination and cultivation could increase risks of infection. Also, in comparison with other prior methods that do not involve germination, the present method affords greater sensitivity.
At present, the method is embodied in a laboratory procedure, though it would be desirable to implement the method by means of a miniaturized apparatus in order to make it convenient and economical enough to encourage widespread use. The main steps of the laboratory procedure (see figure) are the following:
- A sample suspected of containing spores is suspended in an aqueous solution.
- In the first of two heat shocks, the suspension is heated to a temperature of 80 °C for 15 minutes to kill non-spore-forming bacteria and vegetative cells.
- The suspension is subjected to a standard low-acceleration centrifugation wash.
- The cells from the centrifuged suspension are resuspended in a solution that contains adenosine phosphate deaminase, which eliminates extracellular adenosine triphosphate (ATP) and AMP.
- The new suspension is subjected to the aforementioned standard low-acceleration centrifugation wash.
- The cells from the centrifuged suspension are resuspended in distilled water.
- In the second heat shock, the suspension is heated to 100 °C for 10 minutes, causing the release of AMP from any spores that may be present.
- A fraction of the heat-shocked suspension is treated using a commercially available bioluminescence agent with pyruvate, orthophosphate dikinase, causing luminescence in proportion to the concentration(s) of ATP that are converted from AMP by oxidative phosphorylation. The luminescence is measured by use of a standard laboratory luminometer.
- To further ensure that the bioluminescence is of spore (AMP) origin and to discriminate against any residual ATP in the suspension, a remaining untreated fraction of the suspension from step 6 is similarly tested for luminescence that responds to ATP but not AMP.
In experiments on eight strains of Bacillus, this method was found to enable detection of spores in suspension down to sub-femtomolar levels of AMP. These levels correspond to sensitivity of the order of 100 or fewer spores per sample.