**1. Introduction**

In this chapter, the use of nucleic acids in molecular diagnostic testing will be described. Detailed disease area examples will be discussed to illustrate technical capabilities as well as the medical relevance of such testing.

Polymerase chain reaction (PCR) was invented by Kary B. Mullis in the 1980s. Fundamentally, PCR is a cyclic process designed to specifically replicate (amplify) nucleic acid sequences from as little as one to a few strands of DNA. The target DNA is heated to separate double-stranded DNA sequences; short oligonucleotide "primers" that define the portion of the genome to be replicated bind to the target DNA. The primers are extended by a DNA polymerase making a copy of the target DNA. After multiple cycles in which the concentration of the replicated target DNA increases exponentially, the amplified product (amplicon) can be visualized by gel electrophoresis or measured by detection of labeled PCR product (amplicon) by incubation

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with additional reagents to produce color or by fluorescent probe detection. This technique improved the ability to diagnose a number of diseases by enabling identification of many human pathogens that had previously been difficult to detect due to their low concentration in the sample. With the addition of a reverse transcription step to the original PCR process, RNA could be converted to cDNA and then replicated with the PCR process. Thus, the utility of the technique was broadened to detect RNA viruses and eukaryotic mRNA.

A key technical improvement was introduced by Higuchi et al.[4, 5] who developed real-time polymerase chain reaction (RT-PCR), which follows the kinetics of the PCR and detects PCR products during the process of amplification (Figure 1). With RT- PCR, accurate and repro‐ ducible quantitation of pathogen concentration could be incorporated into the amplification process. RT-PCR is used to monitor a pathogen's kinetic replication processes over time, and measurement of viral load is now widely employed to monitor the success of treatment of viral and other diseases.

Figure 1: Principles of Real‐Time PCR �A� During the polymerization step, the template is amplified by primers supplied in the **Figure 1.** Principles of Real-Time PCR. (A) During the polymerization step, the template is amplified by primers sup‐ plied in the reaction mix. The amplicon allows for annealing of sequence-specific, labeled probes. As a new strand is synthesized, the probes will be displaced, the label cleaved off, and a fluorescent signal proportional to the amount of the cleaved probe is generated. (B) Fluorescence is measured and recorded at each cycle of PCR. Cycle threshold (Ct) is defined as the fractional PCR cycle number in which the sample fluorescence signal reaches a level above an assigned fluorescence threshold. The Ct value indicates the beginning of the exponential amplification of the template DNA or RNA and is proportional to the concentration of the sample.[6]

reaction mix. The amplicon allows for annealing of sequence‐specific, labeled probes. As a new strand is synthesized, the probes will be displaced, the label cleaved off, and a fluorescent signal proportional to the amount of the cleaved probe is generated. Clinical microbiology was one of the first fields to adopt PCR and, later, RT-PCR, due to the sensitivity and specificity of the technique for detecting nucleic acids of pathogenic microor‐ ganisms.

�B� Fluorescence is measured and recorded at each cycle of PCR. Cycle threshold �Ct� is defined as the fractional PCR cycle number in which the sample fluorescence signal reaches a level above an assigned fluorescence threshold. The Ct value indicates the beginning of the Perkin-Elmer developed the first thermal cycler instrument in December 1985. The first commercial *in vitro* PCR diagnostic products were created when the California company, Cetus, entered into a partnership with Kodak in February 1986.[7] The first reagent kit, the "Gene-Amp PCR reagent kit" and the thermal cycler were commercially available in Novem‐ ber 1987.[8]

exponential amplification of the template DNA or RNA and is proportional to the concentration 

Clinical microbiology was one of the first fields to adopt PCR and, later, RT‐PCR, due to the 

Perkin‐Elmer developed the first thermal cycler instrument in December 1985. The first 

PCR reagent kit" and the thermal cycler were commercially available in November 1987.8

entered into a partnership with Kodak in February 1986.7 The first reagent kit, the "Gene‐Amp 

 PCR diagnostic products were created when the California company, Cetus, 

sensitivity and specificity of the technique for detecting nucleic acids of pathogenic 

of the sample.6

microorganisms. 

in vitro

commercial 

In January 1989, it was announced that Roche had entered into an agreement with Cetus to develop diagnostic applications for PCR. A new area of molecular diagnostics began using PCR to detect genes and pathogen genomes to diagnose diseases since that time.[7]

At Cetus, it was decided in the late 1980s that the forensic applications of PCR represented a stand-alone business that could be operated in-house, and, therefore, the applications were not sold to a partner.[7] In 1990, the first forensic PCR kit, developed by Cetus, was sold by Perkin-Elmer and became useful for identification of individual humans. The nucleic acid, DNA, became a mainstay of the justice system in 1997 when the FBI announced the selection of 13 short tandem repeat (STR) DNA loci to constitute the core of a national database— Combined DNA Index System (CODIS). By the time a review was published in 2006,[9] 5 million profiles of individuals existed in CODIS. By 2003, almost 1 million samples were being processed annually using core STR loci as part of parentage testing.[10]

Over the past decades, RT-PCR technology has continued to develop, optimize, and expand in the clinical laboratory for the identification, detection, and quantitation of a variety of pathogen uses. Automated instrument platforms were created to facilitate the workflow and allow for accurate and precise processing of patient samples in a highly automated manner.
