The choice of biological molecule for analysis will determine the type of sample preparation; these can involve DNA, RNA, lipids, fatty acids, and proteins either from whole cells or cell extracts. The most abundant of these, however, is protein, representing 50% of the dry weight of the cell; the least abundant is the DNA, with only one copy per cell. Lipids and fatty acids represent 5% to 8% and RNA 0.01%. In most cases, the analyte component is derived from a pure bacterial culture. To access the component of interest, generally the cell is lysed by exposing the cell to water, solvent, and/or strong acid and depending upon the molecule to be analyzed, further extraction, purification, and/or amplification steps may be required. Currently, the most widely used MALDI-TOF MS techniques for bacterial identification are DNA and protein analysis, these are discussed in more detail below.
Identification techniques using DNA/RNA [e.g., single nucleotide polymorphism (SNP)] compare a limited DNA sequence of an unknown bacterium to a database of known sequences. Preparation is similar to other DNA techniques, although in this case the sequence analysis is achieved using MALDI-TOF MS. In this technique, the DNA is extracted and primers are chosen to select a target region (e.g., 16S rDNA), which is amplified and subjected to base-specific cleavage by polymerase chain reaction (PCR), transcription to RNA, followed by a clean-up procedure. The MALDI-TOF MS analysis of the DNA sequence is then determined and used for identification against a database of known sequences, for example, GenBank (Nordhoffet al., 1996; von Wintzingerode et al., 2002; Lefmann et al., 2004).
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