HSV Clinical Specimens, Analyzed by Real-Time PCR Using Extracted and Unextracted Specimens. Clinical specimens found to be positive for HSV-1 (A), HSV-2 (B), or found to be negative for HSV (C) when analyzed by cell culture and immunofluorescence microscopy were analyzed by real-time PCR using nucleic acid-extracted and unextracted samples of each specimen. The amplification curves for the internal controls of the reactions shown in A, B and C are shown in D, E and F respectively. For extracted samples, 200 μl of a clinical specimen (in viral transport buffer) was subjected to automated total nucleic acid extraction with an elution volume of 50 μl; 5 μl of the eluted sample was analyzed. For unextracted samples, 5 μl, 2.5 μl and 1 μl of straight clinical specimen were analyzed. The specimens were taken by swab of genital lesion of a male (A, D) or female (B, C, E, F). The swabs were placed in viral transport buffer and stored frozen (-35°C) until analysis. Probes hybridizing to HSV DNA were detected in the 640 nm channel of a Light Cycler 2.0. Probes specific for internal control DNA were detected in the 705 nm channel. The positive real-time PCR control in Figure 1C was 10,000 copies of a purified plasmid containing HSV-2 DNA target fragment (provided by the real-time PCR kit).