Responsible Interpretation of Polymerase Chain Reaction Assays

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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 un-culturable 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. In a simplified summary, nucleic acid is extracted from a sample; mixed with various reagents; and amplified in 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—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. PCR assays are not flawless, and results can be misinterpreted.

PCR assays are designed to be highly sensitive (do not incorrectly report positive samples as negative) and specific (do not incorrectly report negative samples as positive). Sensitivity and specificity for a given agent can vary between assays and laboratories, because not all assays use the same reagents or amplify the same segment of nucleic acid.

A negative sample can be incorrectly reported as positive for a number of reasons. Samples can be contaminated, related organisms can have similar fingerprints, and nucleic acid can be detected from non-viable organisms. Similarly, positive samples may be incorrectly identified as negative due to inhibitors in the sample, collection of suboptimal samples (e.g., shallow versus deep nasal swabs for herpesvirus), and collection of samples at suboptimal times (e.g., after the period of peak shedding, or after implementation of antimicrobial therapy). These potential problems mandate that PCR results be interpreted in conjunction with appropriate clinical signs.

PCR assays detect nucleic acid and not a disease. Knowledge of the epidemiology for the agent being assayed, the clinical signs and/or pathology induced by the infectious agent, and vaccination history are important for correct interpretation. Certain infectious agents can be normally found throughout the environment or within nonclinical carriers, thus detection of an agent in an animal without clinical disease should be warily interpreted. Additionally, PCR interpretation following recent vaccination with a modified-live vaccine should be carefully interpreted, because these vaccines may induce positive PCR results.

In summary, PCR assays are extremely useful tools to identify infectious agents. However, results should be cautiously interpreted due to the potential variations in assays, techniques, and samples; detection of pathogens in the absence of disease; and vaccine interference.

Contact Dr. Alany Loynachan at alan.loynachan@uky.edu or call 859-257-8283.

Information provided by the University of Kentucky Gluck Equine Research Center’s Lloyd’s Equine Disease Quarterly.