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Nucleic acid amplification technologies are well-characterized methods for the rapid and sensitive diagnosis of a wide variety of infectious diseases. Herpes simplex virus amplification is no exception, demonstrating as much as a ninefold enhancement in sensitivity over viral culture in some settings. Currently, there are no US Food and Drug Administration-approved systems for herpes simplex virus amplification; hence, most laboratories utilize in-house-developed PCR-based systems. Unfortunately, the utilization of herpes simplex virus amplification has been confined to the investigations of suspected herpes simplex virus encephalitis, largely due to cost and the need for appropriately trained technical staff. Recent methodological advances will help to render herpes simplex virus nucleic acid amplification technologies more applicable to routine practice. However, the use of nucleic acid amplification technologies for the diagnosis of genital herpes infection remains highly controversial.