Figure 8-5 Peptide nucleic acid (PNA) probes. Structure of DNA compared lo the structure of a synthetic PNA probe; the chemical modification of DNA allows for greater sensitivity and specificity of the PNA probes compared to the DNA probes. (Courtesy AdvanDx, Woburn, Mass.)
procedures for testing a relatively large number of specimens.
In Situ Hybridization. In situ hybridization allows a pathogen to be identified within the context of the pathologic lesion being produced. This method uses patient cells or tissues as the solid support phase. Tissue specimens thought to be infected with a particular pathogen are processed in a manner that maintains the structural integrity of the tissue and cells yet allows the nucleic arid of the pathogen to be released and denatured to a single strand with the base sequence intact. Although the processing steps required to obtain quality results can be technically difficult, this method is extremely useful because it combines the power of molecular diagnostics with the additional information that histopathologic examination provides.
Peptide Nucleic Acid (PNA) Probes. PNA probes are synthetic pieces of DNA that possess unique chemical characteristics in which the negatively charged sugar-phosphate backbone of DNA is replaced by a neutral polyamide backbone of repetitive units (Figure 8-5). Individual nucleotide bases can be attached to this neutral backbone, which then allows the PNA probe to hybridize to complementary nucleic acid targets. Because of the synthetic structure of the backbone, these probes have improved hybridization characteristics, providing fester and more specific results than traditional DNA probes. In addition, these probes are not degraded by ubiquitous enzymes such as nucleases and proteases, thereby providing longer shelf-life in diagnostic applications, PNA FISH is a novel fluorescence in situ hybridization (FISH) technique using PNA probes targeting species-specific rRNA sequences. Upon penetration of the microbial cell wall, the fluorescent-labeled PNA probes hybridize to multicopy rRNA sequences within the microorganisms, resulting in fluorescent cells. Recently, AdvanDx (Wobum, Massachusetts) introduced in vitro diagnostic kits (employing PNA FISH), cleared by the Food and Drug Administration, to directly identify Staphylococcus aureus and Candida albicans, and to differentiate Bnterococcus faecalis from other enterococd in blood cultures within 2xk hours. In brief, a drop from a positive blood culture bottle is added to a slide containing a drop of fixative solution. After fixation, the fluorescent-labeled PNA probe is added and allowed to hybridize; slides are washed and air-dried. Following the addition of mounting medium and a coverslip, slides are examined under a fluorescent microscope using a spedal filter set. Identification is based on the presence of bright green, fluorescent-staining organisms (Figure 8-6, A and B). For negative results, only slightly red-stained background material is observed (see Figure 8-6, C and 2>). Multiple studies evaluating the $. aureus PNA FISH and C. albicans PNA FISH kits to directly detect S, aureus and C. albicans, respectively, in positive blood cultures have demonstrated high sensitivity and speafidty.
Hybridization with Signal Amplification. To increase the sensitivity of hybridization assays, methods have been developed in which detection of the binding of the probe to its spedfic target is enhanced. For example, one commercially available kit uses genotype-specific RNA probes in either a high-risk or low-risk cocktail to detect human papillomavirus (HFV) DNA in clinical specimens (see Chapter 51). Essentially, sensitivity of HPV detection by hybridization is- increased by multimeric layering of reporter molecules that increases their number on an antibody directed toward DNA-RNA hybrids using chemiluminescence; thus, sensitivity of detection is enhanced by virtue of greater signal produced (i.e„ chemiluminescence) for each antibody bound to target.
Although hybridization methods are highly specific for organism detection and identification, they are limited by their sensitivity, that is, without sufficient target nudeic add in the reaction, false-negative results occur. Therefore, many hybridization methods require "amplifying" target nuddc add by growing target organisms to greater numbers in culture. The requirement for cultivation detracts from the potential speed advantage that molecular methods can offer. Therefore, the devdopment of molecular amplification techniques not dependent on organism multiplication has contributed greatly to circumvent the speed problem while enhancing sensitivity and maintaining spedfidty. For purposes of discussion, amplification methods are divided into two major categories: those
8-6 Using a fluorescent-tagged peptide nucleic acid (PNA) probe in conjunction with fluorescent in situ hybridization (FISH), Staphylococcus aureus (A) or Candida albicans (B) was directly identified in blood cultures within 2V2 hours. A drop from the positive blood culture bottle is added to a slide containing a drop of fixative solution, which keeps the cells intact. After fixation, the appropriate fluorescent-labeled PNA probe is added. The PNA probe penetrates the microbial cell wall and hybridizes to the rRNA. Slides are examined under a fluorescent microscope. If the specific target is present, bright green, fluorescent-staining organisms will be present. Blood cultures negative for either S. aureus <C) or C. albicans (D) by PNA FISH technology are shown. (Courtesy AdvanOx, Wobura, Mass.)
methods that use polymerase chain reaction (PCR) technology and those assays that are not PCR-based.
The most widely used target nucleic acid amplification method is the polymerase chain reaction (PCR). This method combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication that are applied repeatedly through numerous cycles. By this method; a single copy of a nucleic acid target, often undetectable by standard hybridization methods, is multiplied to 107 or more copies within a relatively short period. This provides ample target that can be readily detected by numerous methods.
Figure 8-7 Overview of polymerase chain reaction. The target sequence is denatured to single strands, primers specific for each target strand sequence are added, and DNA polymerase catalyzes the addition of deoxynudeotides to extend and produce new strands complementary to each of the target sequence strands (cycle I). The second cycle begins by both double-stranded products of cycle 1 being denatured and subsequently serving as targets for more primer annealing and extension by DNA polymerase. After 25 to 30 cydes, at least 10' copies of target DNA may be produced. (Modified from Ryan KJ, Champoux JJ, Drew WL, et al: Medical microbiology: an introduction to infectious diseases, Norwalk, Conn, 1994, Appleton & Lange.)
1. Denaturation to single strands
— AGTCCATAGTCCArCCAY 11
H ~ ITCCATCCA
3. Primer extension by J 72° C
action of DNA
Extension of complementary sequences t— AGTCCATC d /AAAOTCCATCCAl — TCAGGTATCAGGTAG/ / TTTCAGGTAGGT ■
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