Procedure for the detection of living cells, with integral and functional cell membranes, by nucleic acid amplification techniques

摘要

The method comprises (1) adding a reaction medium comprising at least two disaggregating agents to a cellular sample, one of them being a non-ionic surfactant or a chelating agent, and the other an anionic surfactant; a phosphate buffer at an appropriate pH; an isotonic solution; and at least one cell metabolism activating substance of the sample cells to activate cell metabolism, (2) add the ethidium monoazide compound (EMA) at a final concentration = 10 {mi} M, (3) allow to incubate the obtained mixture, (4) photoactivate the mixture to activate the EMA, (5) lyse the sample to obtain a mixture of genetic material from dead cells, living cells without membrane metabolism and living cells with active membrane metabolism; and (6) detect by means of a nucleic acid amplification technique, the genetic material of living cells and with active membrane metabolism.