Understanding the reaction balances behind the viability PCR protocols based on photoreactive dyes.

Viability-PCR (vPCR) protocols are mainly based on photo-reactive dyes impermeant to intact cell membranes. The absence of cell barriers allows the reagent's interaction with the genetic material after a short incubation period. By light-induced reaction, DNA becomes the unsuitable mould for the polymerases and thus cannot be amplified and detected by PCR. General rules and consensus exist on critical aspects of successful vPCR protocol development. However, the understanding of the vPCR reaction concerning how much reagent is really effective or the proper amount of light has been poorly studied. The convenience of using 600 times more dye than bases pairs exist suggests that although these dyes are DNA intercalating reagents, many organic molecules can adsorb it. Concerning light, no exact references exist about how much energy is needed to activate the azide group of reagents such as propidium monoazide. Therefore, it cannot be calculated in terms of energy how much light needs a vPCR protocol. The general rule is to provide reagents and energy in excess. This work provides different responses (based on experimental results) to both questions, which can contribute to a better understanding of the theoretical basis of vPCR protocols.

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Déclaration de conflit d'intérêts

Declaration of Competing Interest The authors do not report conflicts of interest relevant to this article. In this work, reagents and equipment manufactured by RPD have been used, but the results are not conditioned by their use. Any expert in the field can reach similar conclusions with their means and those different from those described. Frances Codony is an external consultant to RPD in the field of vPCR.