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Over the past decade, the development of a series of nucleic acid ampli- fication (NAA) technologies has opened new avenues for the detection, identifi- cation, and characterization of pathogenic organisms in diagnostic microbiology (Tang et al., 1997; Jungkind and Kessler, 2002; Yolken, 2002). The promise of these techniques is the replacement of traditional biological amplification of live pathogens by enzymatic amplification of specific nucleic acid sequences. These techniques have reduced the dependency of the clinical microbiology laboratory on culture-based methods and created new opportunities for the field to enhance patient care. According to the theoretical basis for each methods, nucleic acid amplification techniques can be placed into one of three broad categories, which all share certain advantages over traditional methods, particularly for the detection of fastidious, unculturable, and/or highly contagious organisms (Table 10.1). Application of NAA techniques enhances the speed, sensitivity, and sometimes the specificity of an etiologic diagnosis (Tang et al., 1999; Yolken, 2002; Hayden, 2004).

The polymerase chain reaction (PCR) is an technique used to replicate, or amplify, a specific region of DNA billions-fold in just a few hours (Saiki et al., 1985, 1988; Mullis and Faloona, 1987). The amplification is primer directed— oligonucleotide primers anneal to and flank the DNA region to be amplified. PCR is used in diagnostic and research laboratories to generate sufficient quantities of DNA to be adequately tested, analyzed, or manipulated. Because of the exquisite sensitivity it offers, PCR has rapidly become a standard method in diagnostic microbiology. More recently, reagent kits and various instrument platforms have added speed, flexibility, and simplicity (Tang et al., 1997; Fredricks and Relman, 1999; Tang and Persing, 1999). How significant is the contribution of PCR to the field of biomedicine? This question is perhaps best answered by the results of a PubMed search using the key word “PCR” (214,352 hits) or a search using the key words “PCR” and “diagnosis” (74,447 hits).

Today’s development of target-specific and molecular-based therapies relies more than ever upon accurate and sensitive molecular diagnostic technologies. The assessments of therapeutic regimens in areas such as cancer chemotherapy and gene replacement therapy, as well as identification of infectious agents in the diagnostic microbiology laboratory are essential for the success of molecular medicine. The polymerase chain reaction (PCR) (Saiki et al., 1985) has been the workhorse in these areas, and ample evidence can be found throughout this volume. However, the ability of the PCR to amplify as few as a single copy of double-stranded DNA (dsDNA) targets has presented additional challenges in the diagnostic laboratory. The potential for contamination from carry-over product and amplification ofDNA in contaminated matrices, as well as tight patent restrictions concerning the use of PCR, have been some factors in the search for non-PCR–based target amplifi- cation methods. Currently, the alternatives to PCR for nucleic acid amplification involve mainly isothermal transcription-based amplification (ITA) techniques that are based on either bacteriophage RNA polymerases or a group of highly processive DNA polymerases.

Oligonucleotide probes provide a useful tool for the detection of target nucleic acids by the formation of a double helical structure between complementary sequences. The stringent requirements of Watson–Crick base pairing make hybridization extremely specific. However, the detection of target sequence by hybridization is often insensitive due to the limited number of signal molecules that can be labeled on the probe. In general, the analytical sensitivity of probe hybridization is of the order 10 molecules. Therefore, it cannot meet the needs of most clinical diagnostic applications. Many technologies have been developed to improve the detection sensitivity by amplifying the probe sequence bound to the target. All probe amplification technologies are developed based on the recent advancement in molecular biology and the understanding of nucleic acid synthesis (i.e., ligation, polymerization, transcription, digestion/cleavage, etc.).

Several molecular technologies are designed to avoid target amplification so to minimize the possibility of contamination by target amplification products. One of the alternatives to enzymatic amplification of target nucleic acid such as polymerase chain reaction (PCR) is to increase or amplify the signal generated from the probe molecule hybridized to the target nucleic acid sequence, which is referred to as signal amplification. Commonly used signal amplification technologies include branched DNA (bDNA) and hybrid capture (HC) assays. The bDNA method was initially developed by Chiron (Emeryville, CA, USA) and marketed by Bayer Diagnostics (Emeryville, CA, USA), and the hybrid capture method was developed and marketed by Digene Corporation (Gaithersburg, MD, USA).

The need for accurate detection and characterization of nucleic acid targets has prompted the development of a range of methodologies. Highly complex and often expensive techniques, such as oligonucleotide arrays, are being used increasingly. Such methods can be extremely valuable, but issues such as cost, the need for specialized equipment, and a high level of expertise for both the technical and analytical aspects of implementation may limit their use in a clinical setting (Chee et al., 1996; Cheung et al., 1999). Although such systems are certainly effective for gathering large amounts of information and can be extremely useful in the research arena (Khan et al., 1999), their use may be unnecessary if only single PCR target detection is required. The use of real-time molecular product detection methods, largely relying on the principle of fluorescent resonance energy transfer (Chen et al., 1997) (FRET), has also become quite commonplace. These methods are useful for high-throughput diagnostic assays and are amenable to automation.

The advances of technology to determine the nucleotide sequence of DNA have fundamentally changed the field of biological research and medicine. For diagnostic molecular microbiology, the most precise method of identification of a PCR product (amplicon) is to determine its nucleotide sequence. Although it is not always necessary to sequence the entire amplicon for routine diagnostic procedures, DNA sequence has been used to analyze a broad range of PCR products for bacterial identification; for gene mutations related to antimicrobial resistance; for bacterial strain typing and viral genotyping; and so forth. Most of the amplicons of these applications are large (range approximately from 300 base pairs to 1500 base pairs), and the exact nucleotide sequence of the amplicoms are crucial for the results.

The development of molecular-based methodologies over the past two decades has dramatically improved our ability to detect microorganisms in clinical and environmental samples—enabling detection and identification within hours in many cases. However, most of these methods are only capable of monitoring individual or small groups of organisms at a time. Due to the extreme microbial diversity in many environments, such as the human intestine (Eckburg et al., 2005), it is necessary to monitor hundreds to thousands of different microbial populations simultaneously in order to detect all of the organisms of interest as a whole and understand these communities more comprehensively. Microarrays have the unprecedented potential to achieve this objective as specific, sensitive, quantitative, and high-throughput tools for microbial detection, identification, and characterization. Advances in printing technology have enabled the production of microarrays containing thousands to hundreds of thousands of probes. Although microarrays have been primarily developed and used for gene expression profiling of pure cultures of individual organisms, major advances have recently been made in their application to complex environmental samples. This chapter discusses the basis of different microarray formats and their application to issues of clinical interest. Several reviews on microarray technology have recently been published and may provide additional information of interest (Ye et al., 2001; Zhou and Thompson, 2002; Cook and Sayler, 2003; Zhou, 2003; Bodrossy and Sessitsch, 2004; Schadt and Zhou, 2005; Schadt et al., 2005).

Molecular amplification of specific nucleic acid–based targets associated with microbial organisms has advanced our existing tools in infectious diseases diagnosis (Fredricks, 1999; Louie, 2000; Peruski, 2003; Yang, 2004). Laboratory diagnosis of infectious diseases in the past has relied on cultivation of the microorganisms . Hence the viability of the organisms and the laboratory conditions used to mimic the environment ultimately dictates the successfulness of amplification of the intact organism(s) outside of the infected host. Nucleic acid–based technologies allow detection by amplification of specific microbial genetic material irrespective of viability or integrity of the organism (Nissen, 2002; Gulliken, 2004; Mackay, 2004).

The extreme sensitivity of PCR has proved to be one of the strong points of this laboratory technique, making it possible to detect even small numbers of infectious agents present in a clinical sample. It is unfortunate that this strength can also be a weakness. Billions of copies of DNA are produced by PCR, yet only one doublestrandedDNAmolecule is necessary to carry out the reaction. This sets the stage for possible contamination of new reactions with previously produced PCR product.