Bacterial typing: storing and processing of stabilized reference bacteria for polymerase chain reaction without preparing DNA--an example of an automatable procedure.

Abstract

This paper examines the use of bacteria killed on blood-storage paper as templates (ghosts) and takes, as an example, the PCR ribotyping (amplification of the intergenic spacer regions between the 16S and 23S ribosomal RNA genes) of bacteria as described by Kostman et al. (J. Infect. Dis. 171, 204-208, 1995). All procedures have been particularly designed to be compatible with automation. DNA preparation is inappropriate for routine, high-volume sequence amplification from a diversity of microorganism cultures. Blood-storage/processing media provide another way of processing samples for PCR with distinctive aspects of increased safety and ease of automatibility. Blood-storage paper can be used for killing and processing bacteria to DNA-containing ghosts for reliable PCR. From as little as a few microliters of an overnight culture or a reasonably sized, single colony, rapid processing of large sample numbers is possible. FTA blood-storage medium has additional utility in that long-term cataloguing, storage, and processing of paper, loaded with culture, for PCR, is possible via a variety of sample wash sequences. It can be performed at convenience, after collection, and can be delayed indefinitely. using this approach, the repeatability of some PCR-ribotyping methodology of the type used by Kostman et al. (J. Clin. Microbiol. 30, 2084-2087, 1992) was examined as an exercise to demonstrate the practicality of FTA blood-paper usage and to check some basic features of PCR ribotyping. Five strains of Staphylococcus and one strain Escherichia coli were stored and processed on FTA blood paper, the PCR-ribotype patterns were analyzed and found to be the equal of patterns previously seen via additional DNA preparations.