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Adenosine-5'-triphosphate (ATP) is the primary energy carrier in all living organisms, and its detection in living cells represents a well-established approach. ATP-driven bioluminescence (BL) relying on the D-luciferin-luciferase reaction is a bioanalytical tool widely employed for monitoring hygiene and microbial contamination of foods.Here, we report a straightforward method for ATP BL detection using an ATP sensing paper fabricated with an alternative freeze-dry procedure. The assay can be easily implemented in laboratories equipped with (i) freeze-drying, wax printing, and 3D printing technologies and (ii) instrumentation for BL detection such as benchtop luminometers and portable light detectors including a smartphone camera without the need for additional equipment., Elisa Michelini acknowledges support by MIUR and Prima (Partnership for Research and Innovation in the Mediterranean Area) with FEDKITO project. ICN2 is funded by CERCA programme, Generalitat de Catalunya. Grant SEV-2017-0706 funded by MCIN/AEI/10.13039/501100011033.

This method originated due to the need to quickly and sensitively detect Avocado sunblotch viroid (ASBVd) in nursery and field trees in California. Optimum sampling protocols were developed for leaf collection from different sized trees based on size and branching as well as for fruit. An ethanol containing buffered extract from 1 g of ground leaf tissue was used as the source of RNA. The extract was absorbed onto small pieces (disks) of Whatman No. 1 filter paper which were then washed and dried. RNA was eluted from the filter paper using sterile water and used as a template in a standard single-tube RT-PCR reaction. The RNA adsorbed on the filter paper disks was quite stable, and the disks could be stored for over 1 year and shipped worldwide at ambient temperature with no noticeable decline in the quality or quantity of the resulting RT-PCR products. The filter paper capture method was expanded to the detection of other viroids including Potato spindle tuber viroid, Peach latent mosaic viroid, and Chrysanthemum stunt viroid and was tested with some viruses as well with minor modifications of the standard protocol.