Interpreting Polymerase Chain Reaction Assays

Learn about polymerase chain reaction assays, how they work, and how veterinarians interpret their results.
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Polymerase chain reaction (PCR) assays are commonly used in veterinary medicine for the detection of infectious agents. These tests have grown in popularity due to their cost effectiveness, rapid turn-around time, and ability to detect unculturable pathogens. The judicious interpretation of PCR results will remain imperative as new assays are developed and implemented. Familiarity with PCR technology and the organism being assessed is essential for the appropriate interpretation of results.

Nucleic acid, such as deoxyribonucleic acid (DNA), is a hereditary material that serves as a template to propagate proteins that perform essential cellular functions. Nucleic acid sequences are unique to each organism and can be used as a genetic fingerprint to identify a particular organism.

PCR is a complex technique used to amplify a small segment of nucleic acid. Essentially, nucleic acid is extracted from a sample, mixed with various reagents, and amplified in a machine called a thermocycler. If the nucleic acid of interest is present, then thousands to billions of copies will be made. These copies can be detected by gel-based or real-time platforms. Either platform can be used to determine whether a sample is positive or negative—appropriate positive and negative controls are included in every assay. Using gel-based platforms, results are positive or negative and are not quantitative.

Real-time platforms identify each amplified copy with a fluorescent probe, which can be instantaneously (in real time) detected and displayed by a computer. The detection of the organism’s nucleic acid is considered significant once the number of amplified copies meets a statistically determined threshold (termed the CT value). The CT value indicates the number of times that the sample was amplified before it crossed the threshold and allows for quantitation of the original sample; a lower CT value indicates that more target nucleic acid (more organisms) was present in the original sample

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