desired. ELISAs are now highly automated and efforts are underway to commercially develop well-standardized kits containing appropriate controls and materials. d.
Gene Probes Diagnostic bacteriology, virology, and mycology are rapidly adapting molecular biology techniques in addition to classical identification methods to identify organisms. Thus, diagnostic assays utilizing nucleic acid or DNA probes have now been developed for the detection of numerous pathogenic organisms. Prior to employment of a DNA probe, it must first be demonstrated that the DNA probe is highly specific for the targeted organism. For example, a DNA probe for Mycobacterium tuberculosis (Mtb) should not detect other Mycobacteria species. However, it should detect all Mtb isolates. Described below are various types of DNA probes and formats used for the detection of organisms. (1)
Nick-translated DNA Probes An isolated genomic DNA fragment is enzymatically disrupted and some of the DNA bases replaced with highly radioactive DNA bases [Sambrook et al. 1989]. The radioactive probe is now tested for its ability to bind (hybridize) the extracted DNA or RNA from the organism of interest. Prior to hybridization, the targeted DNA or RNA is either fixed to a membrane, microscope slide or resuspended in an aqueous buffer. After hybridization, the unbound DNA probe is then removed and the specimen DNA or RNA analyzed for bound DNA probe. Alternatively, nonradioactive labeling is often employed, but these probes, in general, are less sensitive than use of phosphorus-32 (32P) labeled DNA [Goltz et al. 1990].
(2)
Synthetic Oligomer DNA Probes A short single-stranded DNA segment, usually 20-60 bases in length is designed and chemically synthesized. If the precise DNA sequence of the targeted organism is known, a complementary probe, representing a perfect match to the targeted DNA is designed. However, the precise sequence of the targeted DNA is often not known. Instead, the starting point for probe design is the amino acid sequence. In this situation, because of codon degeneracy, a single probe exhibiting exact complementarity cannot be designed. Thus, an educated guess, based on understanding the genetic code and codon usage, is used to design the probe. This type of probe usually exhibits a high degree of matching, although seldom is a perfect match achieved. Alternatively, a set or "family" of probes is synthesized. These are designed to cover all possible DNA sequences in the targeted organism. The probes are labeled, usually with 32P, prior to hybridization experiments [Sambrook et al. 1989].
(3)
Polymerase Chain Reaction (PCR) First introduced in 1985, the PCR has revolutionized the way DNA analysis is conducted in clinical and research laboratories. Application of the PCR results in the amplification (the in vitro enzymatic synthesis of thousands of copies) of a targeted DNA [Saiki et al. 1985]. Two synthetic, single-stranded DNA segments, usually 18-25 bases in length, are bound to the targeted DNA. These serve as primers and permit the rapid enzymatic amplification of complementary DNA. The method is extremely sensitive and specific. Culturing of the
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