Laboratori d'Aigües de Mataro, carrer Hèl·lade, 17-19, 08304 Mataró, Spain.
Department of Quality Control, Reactivos para Diagnóstico, S.L., Polígono Industrial Mas d'En Cisa, Calle Josep Tura 9 H, 08181 Sentmenat, Barcelona, Spain.
J Microbiol Methods. 2023 Jun;209:106737. doi: 10.1016/j.mimet.2023.106737. Epub 2023 May 12.
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.
活细胞-PCR（vPCR）方案主要基于不透完整细胞膜的光反应性染料。由于不存在细胞屏障，试剂可以在短孵育时间后与遗传物质相互作用。通过光诱导反应，DNA 成为不适合聚合酶的模具，因此不能通过 PCR 进行扩增和检测。关于成功的 vPCR 方案开发的关键方面存在一般规则和共识。然而，对于 vPCR 反应的理解，例如有多少试剂是真正有效的，或者适当的光量是多少，研究得还不够充分。使用比碱基对多 600 倍的染料的便利性表明，尽管这些染料是 DNA 嵌入试剂，但许多有机分子可以吸附它。关于光，没有关于需要多少能量来激活吖啶基团试剂（如吖啶单叠氮化物）的确切参考。因此，不能根据能量来计算 vPCR 方案需要多少光。一般规则是过量提供试剂和能量。这项工作对这两个问题都提供了不同的（基于实验结果的）反应，可以帮助更好地理解 vPCR 方案的理论基础。
