Effective PCR detection of vegetative and dormant bacterial cells due to a unified method for preparation of template DNA encased within cell envelopes

The unified method of template preparation for PCR in the form of DNA covered by permeabilized cell envelopes was used for the cells of different physiological status (vegetative, dormant forms of different types, and nonviable micromummies). The procedure for the preparation of template DNA included one-stage (boiling in a buffer with chaotropic salts) or two-stage (boiling in a buffer with chaotropic salts followed by treatment with proteinase K) sample preparation. The proposed method proved effective for detection of not only vegetative cells but also of the bacillary spores and the cystlike dormant cells (CLC) of non-spore-forming bacteria. For example, the two-stage sample preparation of Bacillus cereus spores resulted in the PCR sensitivity increase up to the detection level of 3-30 spores per sample; the one-stage sample preparation was three orders of magnitude less efficient (10(4) spores per sample). An increase in the sensitivity of PCR detection (4-10-fold) owing to the use of the two-stage sample preparation was shown for bacillary, staphylococcal, and mycobacterial CLC. The possibility of PCR detection of staphylococcal micromummies with irreversibly lost viability, which were therefore undetectable by plating techniques, was also demonstrated. The application of the unified sample preparation method ensuring efficacious PCR detection of bacterial cells, irrespective of their physiological state, may be a promising approach to more complete detection of microbial diversity and the overall insemination of natural substrates.