PCR, principles, advantages and pitfalls: focus in some viral infections of the anterior segment of the eye

Abstract

AbstractGene amplification using polymerase chain reaction (PCR) has become the gold standard for microbiological diagnosis in eye diseases, particularly in those related to viruses. Even if the specificity and the sensibility indexes are much higher than conventional methods, gene amplification may present some limits. For example, uveitis with low grade viral replication may induce false‐negative PCR results while assessment of the immune charge in aqueous may be positive in such cases. For infections of the ocular surface, PCR is usually much more effective than isolation of the virus in cell culture, but the high sensibility may inversely induce false‐positive results. For example, continuous shedding of herpes simplex in tears is known to occur regularly even in asymptomatic patients, which could mask the real diagnosis of any other cause of ocular redness. Even latest technologies, such as real time quantitative PCR, are not completely satisfying since a misuse of this very sensitive technique may also lead to false positive results if effective controls are not systematically used for each assay, due to the self‐fluorescence of the PCR mix when the number of thermal cycles increases. Inversely, vital dies, anesthetics eye drops or non‐optimal tissues purification may lead to false negative results due to the inhibition of the polymerase reaction.